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PTOMAINES,  LEUCOMAINES, 


BACTERIAL  PROTEIDS: 


THE  CHEMICAL  FACTORS  IN  THE  CAUSATION  OK  DISEASE. 


BY 
VICTOR  C.  VAUGHAN,  Ph.D.,  M.D., 

PROFESSOR   OF    HYGIENE   AND    PHYSIOLOGICAL   CHEMISTRY   IN   THE   UNIVERSITY    OF    MICHIGAN, 
AND    DIRECTOR   OF   THE    HYGIENIC   LABORATORY  ; 


FREDERICK  G.  NOVY,  Sc.D.,  M.D., 

ASSISTANT   PROFESSOR   OF  HYGIENE   AND   PHYSIOLOGICAL   CHEMISTRY  IN   THE    UNIVERSITY 
OF   MICHIGAN. 


SECOND  EDITION",  REVISED  AND  ENLARGED. 


PHILADELPHIA: 

LEA   BROTHERS   &   CO., 
18  91. 


1\ 


Entered  according  to  Act  of  Congress  in  the  year  1891,  by 

LEA   BROTHERS    &    CO., 

In  the  Office  of  the  Librarian  of  Congress  at  Washington,  D.  C. 


DOR  NAN.      PRINTER, 
PHILADELPHIA. 


TO 


ALBERT  B.  PRESCOTT,  Ph.D.,  M.D.,  F.C.S., 

DIRECTOR   UF  THE   CHEMICAL   LABORATORY   IN    THE   UNIVERSITY   OF    MICHIGAN, 


THIS    LITTLE    WORK 


IS    RESPECTFULLY    DEDICATED 


AS   A   SLIGHT   TOKEN   OF   THE   HIGH    ESTEEM    IN   WHICH 


HE   IS   HELD   BY   HIS   FORMER   STUDENTS, 


THE  AUTHOES. 


VI  PREFACE    TO    SECOND    EDITION. 

study  has  certainly  become  one  of  great   interest  to  all 
scientific  students  of  medicine. 

In  the  preparation  of  the  present  edition  we  have 
endeavored  to  utilize  the  latest  and  best  information,  and 
we  can  only  express  our  thanks  for  the  encouragement 
which  we  have  received  from  so  many  sources  and  hope 
that  the  present  effort  will  justify  no  censure. 

University  of  Michigan,  September,  1891. 


PREFACE   TO   FIRST   EDITION. 


Within  the  past  ten  years  much  has  been  said  and 
written  concerning  the  basic  substances  formed  during  the 
putrefaction  of  organic  matter,  and  those  which  are  pro- 
duced by  the  normal  tissue-changes  in  the  living  organism. 
Many  investigators  have  given  their  whole  time  and  atten- 
tion to  the  study  of  these  substances,  and  important  discov- 
eries have  been  made  and  much  light  has  been  thrown  upon 
what  have  heretofore  been  considered  problems  in  medical 
science.  To  collect,  arrange,  and  systematize  the  facts 
concerning  ptomaines  and  leucoma'ines  has  been  our  first 
object.  Although  many  short  essays,  some  of  them  of 
great  value,  have  been  written  with  the  above-mentioned 
object  in  view,  the  present  work  may  be  regarded  as  the 
first  attempt  to  make  this  collation  embrace  everything  of 
importance  on  this  subject.  In  endeavoring  to  accomplish 
this  object  we  have  met  with  many  difficulties.  The  original 
reports  of  the  various  investigators  are  scattered  through 
the  pages  of  medical  and  scientific  journals,  transactions  of 
societies,  monographs,  government  reports,  etc.  However, 
with  few  exceptions  we  have  been  able  to  obtain  the  original 


viii  PREFACE    TO    FIRST    EDITION". 

reports,  and  we  think  that  we  have  included  everything  of 
importance  published  up  to  the  present  year  (1888). 

To  the  physician  the  facts  which  have  been  made  known 
concerning  the  putrefactive  and  physiological  alkaloids 
must  be  of  great  value,  and  if  this  little  work  furnishes 
the  means  by  which  members  of  the  profession  may  become 
better  acquainted  with  the  nature  of  those  poisons  which 
are  introduced  from  without,  and  those  which  are  gener- 
ated within  the  body  of  man,  the  object  of  its  authors  will 
be  accomplished. 

University  op  Michigan,  July,  1888. 


CONTENTS. 


PAGE 

Introduction 13 

CHAPTER  I. 

Definition    and    Classification    of  the    Bacterial 

Poisons 15 

CHAPTER  H. 
Historical  Sketch  of  the  Bacterial  Poisons    .        .      22 

CHAPTER  IK. 

Foods  Containing  Bacterial  Poisons  :  Poisonous  Mus- 
sels, Oysters  and  Eels,  Fish,  Sausage,  Harn,  Canned 
Meats,  Cheese,  Milk,  Ice-cream,  Meal  and  Bread    .        .       36 

CHAPTER  IV. 
General  Considerations  of  the  Relation  of  Bac- 
terial Poisons  to  Infectious  Diseases  :   Classifica- 
tion of  Diseases,  How  Germs  Produce  Disease,  Definition 
of  Infectious  Disease,  Koch's  Rules        .         .  .84 

CHAPTER  V. 
The  Bacterial  Poisons  of  some  of  the  Infectious 
Diseases  :  Anthrax,  Asiatic  Cholera,  Tetanus,  Tuber- 
culosis, Diphtheria,  Suppuration,  The  Summer  Diar- 
rhoeas of  Infancy,  Typhoid  Fever,  Swine-plague  (Hog- 
cholera),  Rabbit  Septicaemia,  Pneumonia,  Malignant 
GEdema,  Puerperal  Fever 101 

CHAPTER  VI. 

The  Nature  of  Immunity-giving  Substances  :  Methods 
of  securing  Immunity  ;  Bacterial  Products  which  Favor 
the  Development  of  Infectious  Diseases         .         .         .     146 


X  CONTENTS. 

CHAPTER  VII. 

PAGE 

The  Germicidal  Proteids  of  the  Blood         .        .        .    152 

CHAPTER  VIII. 

Methods  of  Extracting  Ptomaines.  Basic  Impurities  in 
Reagents.  The  Stas-Otto  Method,  Dragendorff 's  Method, 
Brieger's  Method,  The  Methods  of  Gautier  and  Etard. 
Remarks  upon  the  Methods 157 

CHAPTER  IX. 
Methods  of  Isolating  the  Bacterial  Proteids    .        .    171 

CHAPTER  X. 

The  Importance  of  Ptomaines  to  the  Toxicologist. 
Coniine-like  Substances,  Nicotine,  Strychnine,  Mor- 
phine, Atropine,  Digitaline,  Veratrine,  Delphinine,  Col- 
chicine.   Effect  of  Ptomaines  on  Alkaloidal  Reactions   .     174 

CHAPTER  XI. 

Chemistry  of  the  Ptomaines:  Primary  Amines,  Dia- 
mines, etc.,  the  Choline  Group,  other  Oxygen-containing 
Bases,  Undetermined  Ptomaines.     Tables      .        .        .187 

CHAPTER  XII. 

Chemistry  of    the    Leucomaines  :    Uric  Acid  Group, 

Creatinine  Group,  Undetermined  Leucoma'ines.    Tables     280 

CHAPTER  XIII. 
The  Autogenous  Diseases 852 

CHAPTER  XIV. 

Bibliography  :  Ptomaines,  Leucomaines,  Bacterial  Pro- 
teids, Miscellaneous  .        ...         .         .        .     364 


PTOMAINES,  LEUCOMAINES,  AND  BACTERIAL 
PROTEIDS. 


INTRODUCTION. 

It  is  customary  to  divide  bacteria  into  the  parasitic  and 
the  saprophytic.  The  obligate  parasite  can  live  only  on 
living  matter  •  the  obligate  saprophyte  can  live  only  on 
dead  matter.  Since  all  attempts  to  grow  the  bacilli  of 
syphilis  and  leprosy  on  artificial  media  have  failed,  they  are 
probably  obligate  parasites.  True  parasitic  germs  do  not 
prove  speedily  fatal  to  their  hosts,  because  their  continued 
existence  depends  upon  the  continued  existence  of  their  host, 
or  on  their  transference  to  another  host.  Leaving  out  of 
consideration  the  obligate  bacterial  parasites,  about  which 
very  little  is  known  at  best,  the  above  classification  becomes 
of  but  little  importance  to  us  in  a  study  of  the  causal  rela- 
tion of  germs  to  disease,  because  a  given  bacterium  may 
grow  and  multiply  in  one  part  of  the  body,  while  it  is 
unable  to  do  so  in  another ;  or  it  may  thrive  in  one  species 
of  animal,  while  it  finds  the  conditions  unfavorable  in  an- 
other species  ;  or  similar  differences  may  exist  in  individual 
members  of  the  same  species.  Thus,  the  white  rat  is  ordi- 
narily and  naturally  immune  against  the  bacillus  of  anthrax, 
but  if  the  rat  be  exhausted  by  being  kept  on  a  small  tread- 
mill for  some  hours  it  becomes  susceptible  to  anthrax. 
Recognizing  these  facts,  we  propose  that  bacteria  be  divided 
into  the  toxicogenic  and  the  non-toxicogenic.  Since  we 
know  of  no  infectious  disease  in  which  poisons  are  not 
formed,  the  toxicogenic  germs  only  are  of  interest  to  us. 

2 


14  INTKODUCTION. 

In  the  study  of  these  we  mustmofonry  ascertain  the  nature 
of  the  poisons  which  they  produce,  but  must  know  the  con- 
ditions under  which  they  can  multiply  and  elaborate  these 
poisons.  To  these  points  the  following  pages,  in  so  far  as 
they  treat  of  the  infectious  diseases,  will  be  devoted. 

However,  all  diseases  are  not  infectious ;  all  poisons 
formed  within  the  body  do  not  owe  their  existence  to  bac- 
teria. Some  originate  in  the  altered  metabolism  of  the 
various  tissues,  and  these  will  be  discussed  under  the  auto- 
genous diseases. 


CHAPTER   I. 

DEFINITION   AND   CLASSIFICATION   OF  THE   BACTERIAL 
POISONS. 

Ptomaines. — An  exact  classification  of  the  chemical 
factors  in  the  causation  of  the  infectious  diseases  can  prob- 
ably not  be  made  at  present.  We  know  of  two  chemically 
distinct  classes,  one  of  which  contains  substances  which 
combine  with  acids,  forming  chemical  salts,  and  which  in 
this  respect  at  least  correspond  with  the  inorganic  and 
vegetable  bases.  The  members  of  this  class  are  designated 
as  ptomaines,  a  name  suggested  by  the  Italian  toxicologist, 
Selmi,  and  derived  from  the  Greek  word  irrufia,  meaning  a 
cadaver.  A  ptomaine  may  be  defined  as  a  chemical  com- 
pound which  is  basic  in  character  and  which  is  formed  by 
the  action  of  bacteria  on  organic  matter.  On  account  of 
their  basic  properties,  in  which  they  resemble  the  vegetable 
alkaloids,  ptomaines  may  be  called  putrefactive  alkaloids. 
They  have  also  been  called  animal  alkaloids,  but  this  is  a 
misnomer,  because,  in  the  first  place,  some  of  them  are 
formed  in  the  putrefaction  of  vegetable  matter ;  and,  in 
the  second  place,  the  term  "  animal  alkaloid  "  is  more  prop- 
erly restricted  to  the  leucomames — those  basic  substances 
which  result  from  tissue  metabolism  in  the  body.  While 
some  of  the  ptomaines  are  highly  poisonous,  this  is  not  an 
essential  property,  and  others  are  wholly  inert.  Indeed, 
the  greater  number  of  those  which  have  been  isolated  up 
to  the  present  time  do  not,  when  employed  in  single  doses, 
produce  any  apparently  harmful  effects.  Brieger  restricts 
the  term  ptomaine  to  the  non-poisonous  basic  products,  and 
designates  the  poisonous  ones  as  "  toxines."  This  is  a 
classification,  however,  which  seems  to  be  of  questionable 
utility.  It  is  not  always  easy  to  say  just  what  bodies  are 
poisonous  and  what  are  not.     The  poisonous  action  of  a 


16  PTOMAINES. 

substance  depends  upon  the  conditions  under  which,  and 
the  time  during  which,  it  is  administered.  Thirty  grains 
of  quinine  may  be  taken  by  a  healthy  man  during  twenty- 
four  hours  without  any  appreciably,  ill  effect,  yet  few  of  us 
would  be  willing  to  admit  that  the  administration  of  this 
amount  daily  for  three  months  would  be  wise  or  altogether 
free  from  injury.  In  the  same  manner  the  administration 
of  a  given  quantity  of  a  putrefactive  alkaloid  to  a  dog  or 
guinea-pig  in  a  single  dose  may  do  no  harm,  while  the 
daily  production  of  the  same  substance  in  the  intestine  of 
a  man  and  its  absorption  continued  through  weeks  and  pos- 
sibly months  may  be  of  marked  detriment  to  the  health. 
We  do  not  as  yet  know  enough  about  the  physiological  or 
toxicological  action  of  the  putrefactive  alkaloids  to  render 
the  classification  proposed  by  Brieger  worthy  of  general 
adoption. 

All  ptomaines  contain  nitrogen  as  an  essential  part  of 
their  basic  character.  In  this  they  resemble  the  vegetable 
alkaloids.  Some  of  them  contain  oxygen,  while  others  do 
not.  The  latter  correspond  to  the  volatile  vegetable  alka- 
loids, nicotine  and  coniine,  and  the  former  correspond  to 
the  fixed  alkaloids. 

Since  all  putrefaction  is  due  to  the  action  of  bacteria,  it 
follows  that  all  ptomaines  result  from  the  growth  of  these 
micro5rganisms.  The  kind  of  ptomaine  formed  will  de- 
pend upon  the  individual  bacterium  engaged  in  its  produc- 
tion, the  nature  of  the  material  being  acted  upon,  and  the 
conditions  under  which  the  putrefaction  goes  on,  such  as  the 
temperature,  amount  of  oxygen  present,  and  the  duration 
of  the  process. 

Brieger  found  that,  although  the  Eberth  bacillus  grew 
well  in  solutions  of  peptone,  it  did  not  produce  any  pto- 
maine ;  while  from  cultures  of  the  same  bacillus  in  beef-tea 
he  obtained  a  poisonous  alkaloid.  Frrz  found  that  whilst 
the  bacillus  butyricus  produces  by  its  action  on  carbohy- 
drates butyric  acid,  in  glycerin  it  produces  propylic  alcohol, 
and  Morin  has  found  amyl  alcohol  among  the  products  of 
this  germi.  Browjst  has  shown  that  while  the  mycoderma 
aceti  converts  ethylic  alcohol  into  acetic  acid,  it  converts 


DEFINITION.  17 

propylic  alcohol  into  propionic  acid,  and  is  without  effect 
upon  methylic  alcohol,  primary  isobutylic  alcohol,  and 
amylic  alcohol.  Some  bacteria  will  not  multiply  below  a 
given  temperature.  Thus,  the  bacillus  butyricus  will  not 
grow  at  a  temperature  below  240.1  The  lower  temperature 
does  not  destroy  the  organism,  but  it  lies  dormant  until  the 
conditions  are  more  favorable  for  its  growth.  Pasteur 
divided  the  bacteria  into  two  classes — the  aerobic  and  the 
anaerobic,  (fe.s  the  name  implies,  the  former  grow  and 
thrive  in  the  presence  of  air,  while  the  latter  find  their 
conditions  of  life  improved  by  the  exclusion  of  air.  There- 
fore, different  ptomaines  will  be  formed  in  decomposing 
matter  freely  exposed  to  the  air,  and  in  that  which  is  buried 
beneath  the  soil  or  from  which  the  air  is  largely  excluded. 
Even  when  the  same  ferment  is  present  the  products  of  the 
putrefaction  will  vary,  within  certain  limits,  according  to 
the  extent  to  which  the  putrefying  material  is  supplied  with 
air.  The  kind  of  ptomaine  found  in  a  given  putrid  sub- 
stance will  depend  also  upon  the  stage  of  the  putrefaction. 
Ptomaines  are  transition  products  in  the  process  of  putre- 
faction. They  are  temporary  forms  through  which  matter 
passes  while  it  is  being  transformed,  by  the  activity  of  bac- 
terial life,  from  the  organic  to  the  inorganic  state.  Com- 
plex organic  substances,  as  muscle  and  brain,  are  broken 
up  into  less  complex  molecules,  and  so  the  process  of 
chemical  division  goes  on  until  the  simple  and  well-knoAvn 
final  products,  carbonic  acid  gas,  ammonia,  and  water, 
result ;  but  the  variety  of  combinations  into  which  an 
individual  atom  of  carbon  may  enter  during  this  long 
series  of  changes  is  almost  unlimited,  and  with  each  change 
in  combination  there  is  more  or  less  change  in  nature.  In 
one  combination  the  atom  of  carbon  may  exist  as  a  con- 
stituent of  a  highly  poisonous  substance,  while  the  next 
combination  into  which  it  enters  may  be  wholly  inert. 

It  was  formerly  supposed  that  putrefaction  was  simply 
oxidatiou,  but  the  researches  of  Pasteur  and  others  have 
demonstrated  the  fact  that  countless   myriads  of  minute 

1  All  temperatures  given  in  this  work  are  Centigrade,  unless  otherwise 
specified. 


18  BACTERIAL    PROTEIDS. 

organisms  are  engaged  constantly  in  transforming  matter 
from  the  organic  to  the  inorganic  form.  Lock  up  the  bit 
of  flesh  so  that  these  little  workers  cannot  reach  it,  and  it 
will  remain  unchanged  indefinitely. 

It  may  be  asked  if  any  of  the  changes  occurring  during 
putrefaction  are  to  be  regarded  as  purely  chemical.  Without 
doubt,  many  of  the  secondary  products  of  putrefaction  arise 
from  reactions  between  antecedent  and  more  complex  prod- 
ucts or  by  the  action  of  oxygen,  water,  and  reducing  agents 
upon  primary  products.  Ptomaines  formed  in  this  way 
may  be  regarded  as  the  indirect  results  of  bacterial  life. 

Bacterial  Proteids. — These  substances  have  been 
known  for  so  short  a  time  and  are  at  present  so  imperfectly 
known  that  many  difficulties  arise  in  discussing  them.  In 
the  first  place,  we  may  divide  the  bacterial  proteids  into 
two  classes  :  (1)  those  which  constitute  an  integral  part  of 
the  bacterial  cells,  and  (2)  those  which  have  not  been 
assimilated  by  the  cells,  but  which  have  been  formed  by 
the  fermentative  or  cleavage  action  of  the  bacteria  on  the 
proteid  bodies  in  which  they  are  growing.  Even  this 
classification  is  of  questionable  value.  We  allow  bacteria 
to  grow  for  a  number  of  days  in  a  nutrient  solution.  We 
then  separate  the  soluble  constituents  from  the  formed  cells 
by  filtration  through  porous  tile ;  we  wash  the  latter  and 
then  study  their  proteid  contents,  which  constitute  the  first 
class,  as  given  above  ;  but  the  filtrate  contains,  or  may  con- 
tain, any  one  or  more  of  the  following  proteid  bodies  :  (1) 
Those  portions  of  the  proteid  substances  which  were  used 
in  the  preparation  of  the  nutrient  solution  and  which  have 
escaped  the  action  of  the  bateria  •  (2)  proteids  which  have 
been  at  one  time  integral  parts  of  the  cells,  but  which 
have  passed  into  solution  on  the  death  and  dissolution  of 
the  bacteria  ;  and  (3)  proteids  which  have  been  formed  by 
the  fermentative  action  of  the  bacteria,  or  those  which  arc 
defined  as  constituting  the  second  class,  as  given  above. 
We  know  at  present  of  no  means  by  which  one  of  these 
proteids  can  with  certainty  be  isolated  from  the  others. 
However,  the  above  classification  is  a  convenient  one,  and 


BACTERIAL    PROTEIDS.  19 

with  a  clear  understanding  that  it  is  not  free  from  criticism 
we  may  employ  it  until  a  more  thorough  and  scientific  study 
of  these  bodies  has  been  made. 

The  difficulty  in  discussing  these  substances  lies  not  only 
in  the  classification,  but  in  the  name  which  shall  be  em- 
ployed to  designate  them.  Brieger  and  Frankel  have 
proposed  the  term  "  toxalbumins  f  but,  while  it  is  true 
that  some  belong  to  the  albumins,  others  are  more  truly 
albumoses  ;  others  are  most  closely  related  to  the  peptones  ; 
and  still  others  differ  in  some  important  respects  from  all 
of  these.  In  view  of  the  above  facts,  we  have  decided  upon 
the  term  "  bacterial  proteids  "  to  designate  those  formed  by 
the  fermentative  action  of  germs,  while  those  which  consti- 
tute an  integral  part  of  the  cell  will  be  known  as  "the 
bacterial  cellular  proteids." 

The  Bacterial  Cellular  Proteids. — Nencki  first  prepared 
one  of  these  substances  from  'putrefactive  bacteria.  These 
were  obtained  by  decantation,  freed  from  fat  with  ether, 
dissolved  in  fifty  parts  of  a  potash  solution  of  0.5  percent., 
heated  for  some  hours  at  100°  and  filtered.  The  filtrate 
was  acidified  with  dilute  hydrochloric  acid  and  precipitated 
by  the  addition  of  rock  salt.  The  precipitate  was  washed 
with  a  saturated  salt  solution,  dried  at  100°,  and  washed 
free  from  salt  with  water.  Nencki  designates  this  sub- 
stance as  "  mycoprotein,"  and  finds  that  it  has  the  formula, 
C25H42N6Og.  Freshly  precipitated  mycoprotein  forms  in 
amorphous  flakes,  which  are  soluble  in  water,  alkalies,  and 
acids.  The  aqueous  solution  is  acid  in  reaction.  After 
being  dried  at  100°  it  is  no  longer  wholly  soluble  in  water. 
Nencki  found  that  it  is  not  precipitated  from  aqueous  solu- 
tion by  alcohol,  but  by  picric  acid,  tannic  acid,  and  mercuric 
chloride ;  that  it  does  not  give  the  xanthoproteid,.  but 
does  give  the  Millon  and  the  biuret  reactions.  According 
to  Schaffer  it  is  changed  by  acids  into  peptone,  and  on 
being  fused  with  five  parts  of  potash  it  breaks  up  into  am- 
monia, amylamin,  phenol  (0.15  per  cent,  of  its  weight),  vale- 
rianic acid  (38  per  cent.),  leucine,  and  traces  of  indol  and 
skatol.  A  proteid  obtained  from  the  yeast  plant  has  the 
formula,  C12H21N303. 


20  BACTERIAL    PROTEIDS. 

The  purified  pyogenetic  agent  obtained  from  the  pneu- 
monia bacillus  of  Friedlander  was  found  by  Buchner 
to  give  the  following  reactions  :  It  is  soluble  in  water  and 
the  concentrated  mineral  acids,  very  soluble  in  dilute  alka- 
lies, from  which  it  is  precipitated  on  the  addition  of  an  acid. 
From  its  aqueous  solution,  it  is  not  precipitated  by  heat, 
nor  by  saturation  with  sodium  chloride,  but  is  precipitated 
by  magnesium  sulphate,  copper  sulphate,  platinum  chloride, 
gold  chloride,  lead  salts,  picric  acid,  tannic  acid,  and  abso- 
lute alcohol.  It  gives  the  xanthoproteid,  Millon,  and 
biuret  reactions. 

The  Bacterial  Proteids. — Brieger  and  Frankel  ob- 
tained the  proteid  poison  of  diphtheria  by  precipitating 
the  filtrate  from  a  Chamberland  filter  after  concentration 
to  one- third  its  volume  at  30°,  with  absolute  alcohol  after 
feebly  acidifying  with  acetic  acid.  The  precipitate  was  puri- 
fied by  repeated  solution  in  water  and  reprecipitation  with 
alcohol.  Dried  in  a  vacuum  at  40°,  it  forms  a  snow-white, 
amorphous,  very  light  mass.  From  its  aqueous  solution  it 
is  not  precipitated  by  heat  or  dilute  nitric  acid,  singly  or 
combined,  nor  by  sodium  sulphate,  sodium  chloride,  mag- 
nesium sulphate,  or  lead 'salts.  It  is  precipitated  by  car- 
bonic acid  (to  saturation),  concentrated  mineral  acids, 
potassium  ferrocyanide  and  acetic  acid,  phenol,  organic 
acids  (soluble  in  excess),  copper  sulphate,  silver  nitrate, 
and  mercuric  chloride.  The  so-called  alkaloidal  reagents, 
phosphomolybdic  acid,  potassio-mecuric  iodide,  potassio- 
bismuthic  iodide,  platinum  chloride,  gold  chloride,  and 
picric  acid  also  cause  precipitation.  The  xanthoproteid, 
Millon,  and  biuret  reactions  give  positive  results.  An  ulti- 
mate analysis  furnishes  the  following  figures  computed 
from  the  ash-free  substance :  C  45.35,  H  7.13,  N  16.33,  S 
1.39,  O  29.80.  From  these  facts  Brieger  and  Frankee 
conclude  that  this  substance  is  allied  to  serum-albumin. 
Their  bouillon  cultures  contain  serum-albumin,  and  they 
suppose  that  the  bacteria  convert  this  into  the  poison  by 
causing  a  rearrangement  in  the  atoms ;  but  the  same  poison 
was  formed  when  nutrient  solutions  containing  no  proteid 


BACTERIAL    PROTEIDS.  21 

save  peptone  were  employed.  In  this  case  they  suppose 
that  the  bacteria  reconvert  the  peptone  into  an  albumin. 

The  poisonous  proteids  obtained  by  Brieger  and 
Frankel  from  cultures  of  the  Eberth  germ,  the  comma 
bacillus,  and  the  staphylococcus  aureus  are  practically  in- 
soluble in  water,  and  more  nearly  related  to  the  globulins 
than  the  albumins,  although  they  differ  from  the  former 
in  their  tardy  and  difficult  solubility  in  dilute  solutions  of 
sodium  chloride. 

The  poisonous  proteids  isolated  by  Vaughan  from  cul- 
tures of  two  species  of  toxicogenic  germs  found  in  drinking 
water,  supposed  to  be  the  cause  of  typhoid  fever,  are  solu- 
ble in  water,  from  which  they  are  not  precipitated  by  boil- 
ing, or  by  concentrated  nitric  acid,  or  by  both.  Potassium 
ferrocyanide  and  acetic  acid,  sodium  sulphate,  magnesium 
sulphate,  and  carbonic  acid  also  fail  to  precipitate  them. 
They  are  precipitated  by  the  general  alkaloidal  reagents, 
and  respond  to  the  xanthoproteid,  Millon,  and  biuret  tests. 
They  are  precipitated  by  ammonium  sulphate  when  added 
to  saturation,  and  for  this  reason  cannot  be  classed  among 
the  peptones.  Neither  benzoyl  chloride  nor  phenyl-hydra- 
zin  chloride  precipitate  them.  Their  poisonous  properties 
are  destroyed  by  prolonged  boiling  or  by  being  heated  to 
80°  for  some  hours,  though  they  remain  active  after  an 
exposure  of  ten  minutes  to  the  last  mentioned  temperature. 

Of  the  three  bacterial  proteids  obtained  by  the  same  ex- 
perimenter from  the  bacilli  x,  a  and  A  of  Booker's  list  of 
summer  diarrhoea  germs,  the  first  two  are  soluble  in  water, 
while  the  other  is  not.  So  far  as  their  behavior  with  pre- 
cipitating agents  is  concerned,  the  first  two  agree  with  the 
proteids  of  the  water  germs. 

Tizzoni  and  Cattani  find  that  the  proteid  of  cultures 
of  their  tetanus  germ  is  rendered  inert  by  precipitation  with 
absolute  alcohol.  It  is  obtained  by  saturation  with  am- 
monium sulphate,  and  the  removal  of  the  salt  by  dialysis. 

Further  description  of  the  individual  proteids  will  be 
given  in  subsequent  chapters. 

2* 


CHAPTER   II. 

HISTORICAL,   SKETCH    OF   THE    BACTERIAL    POISONS. 

It  must  have  been  known  to  primitive  man  that  the 
eating  of  putrid  flesh  was  liable  to  affect  the  health  more 
or  less  seriously ;  and  when  he  began  his  endeavors  to 
preserve  his  food  for  further  use,  instances  of  poisoning 
from  putrefaction  must  have  multiplied.  However,  the 
distinguished  physiologist,  Albert  von  Haller,  seems 
to  have  been  the  first  to  make  any  scientific  experiments 
concerning  the  effects  of  putrid  matter  upon  animals.  He 
injected  aqueous  extracts  of  putrid  material  into  the  veins 
and  found  that  death  resulted.  Later  in  the  eighteenth 
century  Morand  gave  an  account  of  the  symptoms  in- 
duced by  eating  poisonous  meat.  In  the  early  part  of 
the  present  century  (1808  to  1814)  Gaspard  carried  on 
similar  experiments.  He  use  as  material  the  putrid  flesh 
of  both  carnivorous  and  herbivorous  animals.  With  these 
he  induced  marked  nervous  disturbances,  as  stiffness  of  the 
limbs,  opisthotonos,  and  tetanus.  Gaspard  concluded  from 
the  symptoms  that  the  poisonous  effects  were  not  due  to 
carbonic  acid  gas  or  hydrogen  sulphide,  but  thought  it 
possible  that  ammonia  might  have  part  in  their  produc- 
tion. In  1820  Kerner  published  his  first  essay  on  poi- 
sonous sausage,  which  was  followed  by  a  second  in  1822. 
At  first  he  thought  that  the  poisonous  properties  were  due 
to  a  fatty  acid,  similar  to  the  sebacic  of  Thenard,  and 
which  originated  during  putrefaction.  Later  he  modified 
these  views,  and  believed  the  poison  to  be  a  compound  con- 
sisting of  the  sebacic  acid  and  a  volatile  principle.  This  may 
be  regarded  as  the  first  suggestion  as  to  the  probability  of 
the  development  of  a  poisonous  substance  with  basic  prop- 
erties in  decomposing  matter.  In  1822,  Dupre  observed 
a  peculiar  disease  among  the  soldiers  under  his  care,  who, 


HISTORICAL    SKETCH.  23 

during  the  warm  and  dry  summer  of  that  year,  were 
compelled  to  drink  very  foul  water.  Later  Magendie, 
induced  by  the  investigations  of  Gaspard  and  the  obser- 
vations of  Dupre,  made  many  experiments,  in  which  dogs 
and  other  animals  were  confined  over  vessels  containing 
putrid  animal  matter  and  compelled  constantly  to  breathe 
the  emanations  therefrom.  The  effects  varied  markedly 
with  the  species  of  animal  and  the  nature  of  the  putrid 
material,  but  in  some  instances  symptoms  were  induced 
which  resembled  closely  those  of  typhoid  fever  in  man. 
Leuret  directed  his  attention  to  the  chemical  changes 
produced  in  blood  by  putrefaction,  but  accomplished  noth- 
ing of  special  value.  Dupuy  injected  putrid  material  into 
the  jugular  vein  of  a  horse,  and  with  Trousseau  studied 
alterations  produced  in  the  blood  by  these  injections. 

During  the  third  decade  of  the  present  century  there 
were  many  investigators  in  addition  to  those  mentioned 
above,  who  endeavored  to  ascertain  the  active  agent  in 
poisonous  foods.  Dann,  Weiss,  Buchner,  Schumann, 
Cadet  de  Gassicourt,  and  Orfila  studied  poisonous 
sausage,  but  made  no  advance  upon  the  work  done  by 
Kerner.  Henneman,  Hunnefeld,  Westrumb,  and 
Serturner  made  contributions  concerning  poisonous 
cheese,  but  all  believed  the  caseic  acid  of  Kerner  to 
be  the  poisonous  principle. 

In  1850  Schmidt,  of  Dorpat,  made  some  investigations 
on  the  decomposition  products  and  volatile  substances 
found  in  cholera  stools ;  and,  two  years  later,  Meyer,  of 
Berlin,  injected  the  blood  and  stools  of  cholera  patients 
into  lower  animals.  In  1853  Stich  made  an  important 
contribution  on  the  effects  of  acute  poisoning  with  putrid 
material.  He  ascertained  that,  when  given  in  sufficient 
quantity,  putrid  matter  produces  an  intestinal  catarrh,  with 
choleraic  stools.  Nervous  symptoms,  trembling,  unsteady 
gait,  and,  finally,  convulsions  were  also  observed.  Stich 
made  careful  postmortem  examinations,  and  was  unable 
to  find  any  characteristic  or  important  lesions.  Theo- 
retically, he  concluded  that  the  putrid  material  contained 
a  ferment  which  produced  rapid  decomposition  of  the  blood. 


24  BACTERIAL    POISONS. 

In  1856  Panum  published  a  most  important  contribu- 
tion to  the  knowledge  of  the  nature  of  the  poison  present 
in  -putrid  flesh.  He  first  demonstrated  positively  the 
chemical  character  of  the  poison,  inasmuch  as  he  showed 
that  the  aqueous  extract  of  the  putrid  material  retained 
its  poisonous  properties  after  treatment  which  would  insure 
the  destruction  of  all  organisms.  His  conclusions  were  as 
follows : 

(1)  "  The  putrid  poison  contained  in  the  decomposed 
flesh  of  the  dog,  and  which  is  obtained  by  extraction  with 
distilled  water  and  repeated  filtration,  is  not  volatile,  but 
fixed.  It  does  not  pass  over  on  distillation,  but  remains  in 
the  retort. 

(2)  "  The  putrid  poison  is  not  destroyed  by  boiling,  nor 
by  evaporation.  It  preserves  its  poisonous  properties  even 
after  the  boiling  has  been  continued  for  eleven  hours,  and 
after  the  evaporation  has  been  carried  to  complete  desicca- 
tion at  100°. 

(3)  "  The  putrid  poison  is  insoluble  in  absolute  alcohol, 
but  is  soluble  in  water,  and  is  contained  in  the  aqueous  ex- 
tract which  is  formed  by  treating  with  distilled  water  the 
putrid  material  which  has  previously  been  dried  by  heat 
and  washed  with  alcohol. 

(4)  "  The  albuminoid  substances  which  frequently  are 
found  in  putrid  fluids  are  not  in  themselves  poisonous  only 
so  far  as  they  contain  the  putrid  poison  fixed  and  condensed 
upon  their  surfaces,  from  which  it  can  be  removed  by 
repeated  and  careful  washing. 

(5)  "The  intensity  of  the  putrid  poison  is  comparable 
to  that  of  the  venom  of  serpents,  of  curare,  and  of  certain 
vegetable  alkaloids,  inasmuch  as  0.012  of  a  gramme  of  the 
poison,  obtained  by  extracting  with  distilled  water  putrid 
material  which  had  been  previously  boiled  for  a  long  time, 
dried  at  100°,  and  submitted  to  the  action  of  absolute 
alcohol,  was  sufficient  almost  to  kill  a  small  dog." 

Panum  made  intravenous  injections  with  this  poison,  and 
with  ammonium  carbonate,  ammonium  butyrate,  ammo- 
nium valerianate,  tyrosine,  and  leucine,  and  found  that  the 
symptoms  induced  by  the  putrid  poison  differed  from  those 


HISTORICAL -SKETCH.  25 

caused  by  the  other  agents.  Moreover,  he  found  the  symp- 
toms to  differ  from  those  of  typhoid  fever,  cholera,  pyaemia, 
anthrax,  and  sausage  poisoning.  He  was  also  in  doubt  as  to 
whether  the  poison  acted  directly  upon  the  nervous  system, 
or  whether  it  acted  as  a  ferment  upon  the  blood,  causing 
decomposition,  the  products  of  which  affected  the  nerve- 
centres  ;  but  he  was  sure  that  it  could  not  correspond  to 
the  ordinary  ferments,  inasmuch  as  it  was  not  decomposed 
by  prolonged  boiling  nor  by  treatment  with  absolute  alcohol. 
Certainly,  the  putrid  poison  could  not  consist  of  a  living 
organism. 

The  symptoms  observed  by  Pajstum  varied  greatly  with 
the  quantity  of  the  poison  used  and  the  strength  of  the 
animal.  After  the  intravenous  injection  of  large  doses, 
death  followed  in  a  very  short  time.  In  these  cases  there 
were  violent  cramps,  and  involuntary  evacuations  of  the 
urine  and  feces ;  the  respirations  were  labored,  the  pallor 
was  marked,  sometimes  followed  by  cyanosis,  the  pulse 
feeble,  the  pupils  widely  dilated,  and  the  eyes  projecting. 
In  these  cases  the  autopsy  did  not  reveal  any  lesion,  save 
that  the  blood  was  dark,  imperfectly  coagulated  and  slightly 
infiltrated  through  the  tissue.  Post-mortem  putrefaction 
came  on  with  extraordinary  rapidity. 

When  smaller  doses  or  more  vigorous  animals  were  used, 
the  symj)toms  did  not  appear  before  from  a  quarter  of  an 
hour  to  two  hours,  and  sometimes  even  later.  In  these 
cases  the  symptoms  were  less  violent,  and  the  animal  gen- 
erally recovered.  In  all  instances,  however,  the  disturbances 
were  more  or  less  marked. 

In  addition  to  the  "  putrid  poison,"  Panum  obtained  a 
narcotic  substance,  the  two  being  separated  by  the  solubility 
of  the  narcotic  in  alcohol.  The  alcoholic  extract  was  evap- 
orated to  dryness,  the  residue  dissolved  in  water  and  injected 
into  the  jugular  vein  of  a  dog.  The  animal  fell  into  a  deep 
sleep,  which  remained  unbroken  for  twenty-four  hours, 
when  it  awoke  apparently  in  perfect  health. 

Pantjm's  first  contributions,  which  were  published  in 
Danish,  did  not  attract  the  attention  which  they  deserved, 
until  after  the  lapse  of  several  years.     Now,  however,  their 


26  BACTERIAL    POISONS. 

importance  is  fully  appreciated,  and  the  distinguished  inves- 
tigator lived  to  receive  the  credit  and  honor  due  him. 

Weber,  in  1864,  and  Hemmer  and  Schwenninger 
in  1866,  confirmed  the  results  obtained  by  Panum  ;  and 
Schwenninger  announced  that  in  the  various  stages  of 
putrefaction  different  products  are  formed,  and  that  these 
vary  in  their  effects  upon  animals.  In  1866,  Bence 
Jones  and  Dupre  obtained  from  the  liver  a  substance 
which  in  solutions  of  dilute  sulphuric  acid  gives  the  blue 
fluorescence  observed  in  similar  solutions  of  quinine.  To 
this  substance  they  gave  the  name  "  animal  chinoidine." 
Subsequently,  the  same  investigators  found  this  substance 
in  all  organs  and  tissues  of  the  body,  but  most  abundantly 
in  the  nerves.  Its  feebly  acid  solutions  give  precipitates 
with  iodine,  potassio-mercuric  iodide,  phospho-molybdic 
acid,  gold  chloride,  and  platinum  chloride.  From  three 
pounds  of  sheep's  liver,  they  obtained  three  grammes  of  a 
solution  in  which,  after  slight  acidulation  witli  sulphuric 
acid,  the  intensity  of  the  fluorescence  was  about  the  same 
as  that  of  a  similarly  acidulated  solution  of  quinine  sulphate 
which  contained  0.2  gramme  of  quinine  per  litre.  Still 
later,  this  base  was  obtained  by  Marino-Zuco. 

In  1868,  Bergmann  and  Schmiedeberg  separated, 
first  from  putrid  yeast,  and  subsequently  from  decomposed 
blood,  in  the  form  of  a  sulphate,  a  poisonous  substance 
which  they  named  sepsine.  The  sulphate  of  sepsine  forms 
in  needle-shaped  crystals.  Small  doses  (0.01  gramme)  of 
this  substance  were  dissolved  in  water  and  injected  into  the 
veins  of  two  dogs.  In  a  short  time  it  produced  vomiting, 
and  later  diarrhoea,  which,  in  one  of  the  animals,  after  a 
time,  became  bloody.  Post-mortem  examination  showed, 
in  the  stomach  and  intestines,  bloody  ecchymoses.  It  was 
now  believed  that  the  "putrid  poison"  of  Panum  had  been 
isolated,  and  that  it  was  identical  with  sepsine,  but  further 
investigations  showed  that  this  was  not  true.  There  are 
marked  differences  in  their  effects  upon  animals,  and  sepsine 
has  not  been  found  to  be  generally  j>resent  in  putrid  ma- 
terial. It  is  only  rarely  found  in  blood,  and  the  closest 
search  has  failed  to  show  its  presence  in  pus.    Bergmann, 


HISTORICAL    SKETCH.  27 

following  the  same  method  which  he  had  used  in  extracting 
this  poison  from  yeast,  has  been  unable  to  obtain  it  from 
other  putrid  material.  Moreover,  he  was  not  always  suc- 
cessful in  obtaining  the  poison  from  yeast.  Sepsine  was 
not  obtained  in  quantity  sufficient  to  serve  for  an  ultimate 
analysis,  hence,  its  composition  remains  unknown. 

In  1869  Zulzer  and  Sonnenschein  prepared  from 
decomposed  meat  extracts  a  nitrogenous  base,  which  in  its 
chemical  reactions  and  physiological  effects  resembled  atro- 
pine and  hyoscyamine.  When  injected  under  the  skin  of 
animals  it  produced  dilatation  of  the  pupils,  paralysis  of 
the  muscles  of  the  intestines,  and  acceleration  of  the  heart- 
beat ;  but  it  is  uncertain  and  inconstant  in  its  action.  This 
probably  results  from  rapid  decomposition  taking  place  in 
it,  or  to  variations  in  its  composition  at  different  stages  of 
putrefaction.  This  substance  has  also  been  obtained  from 
the  bodies  of  those  who  have  died  from  typhoid  fever,  and 
it  may  be  possible  that  the  belladonna-like  delirium  which 
frequently  characterizes  the  later  stages  of  this  disease  is 
due  to  the  ante-mortem  generation  of  this  poison  within 
the  body. 

Since  1870  many  chemists  have  been  engaged  in  making 
investigations  on  the  products  of  putrefaction.  We  can 
only  mention  a  few  names  at  present,  while  others  will  be 
referred  to  subsequently  in  discussing  the  individual  pto- 
maines. 

First  of  all  stands  the  Italian  Selmi,  who  suggested  the 
name  ptomaine,  and  whose  researches  furnished  us  with 
much  information  of  value,  and,  what  is  probably  of  more 
importance,  gave  an  impetus  to  the  study  of  the  chemistry 
of  putrefaction,  which  has  already  been  productive  of  much 
good  and  gives  promise  of  much  more  in  the  future.  Selmi 
showed  that  ptomaines  could  be  obtained  (1)  by  extracting 
acidified  solutions  of  putrid  material  with  ether ;  (2)  by 
extracting  alkaline  solutions  with  ether ;  (3)  by  extracting 
alkaline  solutions  with  chloroform  ;  (4)  by  extracting  with 
amylic  alcohol ;  and  (5)  that  there  yet  remained  in  the  solu- 
tions of  putrid  matter  ptomaines  which  were  not  extracted 
by  any  of  the  above-mentioned  reagents.     In  this  way  he 


28  BACTEKIAL    POISONS. 

gave  some  idea  of  the  great  number  of  alkaloiclal  bodies 
which  might  be  formed  among  the  products  of  putrefaction, 
and  the  promising  field  thus  discovered  and  outlined  was 
soon  occupied  by  a  busy  host  of  chemists.  In  the  second 
place,  he  demonstrated  the  fact  that  many  of  the  ptomaines 
give  reactions  similar  to  those  given  by  the  vegetable  alka- 
loids. This  led  the  toxicologist  into  investigations,  the 
results  of  some  of  which  we  will  ascertain  further  on. 

Selmi,  however,  did  not  succeed  in  isolating  completely 
a  single  putrefactive  alkaloid.  All  his  work  was  done  with 
extracts.  He  remained  ignorant,  except  in  a  general  way, 
of  the  composition  of  these  bodies.  Nencki,  in  1876, 
made  the  first  ultimate  analysis  and  determined  the  first 
formula  of  a  ptomaine.  This  was  an  isomer  of  collodine, 
which  will  be  described  later. 

Rorsch  and  Fassbender,  in  a  case  of  suspected  poison- 
ing, obtained  by  the  Stas-Otto  method  a  liquid  which 
could  be  extracted  from  acid  as  well  as  alkaline  solutions 
by  ether,  and  which  gave  all  the  general  alkaloidal  reac- 
tions. They  were  unable  to  crystallize  either  extract  by 
taking  it  up  with  alcohol  and  evaporating.  The  colorless 
aqueous  solution  was  not  at  all  bitter  to  the  taste.  The 
precipitate  formed  with  phospho-molybdic  acid  dissolved  on 
the  application  of  heat,  giving  a  green  solution,  which 
became  blue  on  the  addition  of  ammonia.  They  believed 
that  this  substance  was  derived  from  the  liver,  since  fresh 
ox-liver,  treated  in  the  same  manner,  gave  them  an  alkaloid 
which  could  be  extracted  with  ether  from  acid  as  well  as 
from  alkaline  solutions.  Gunning  found  this  same  alka- 
loid in  liver-sausage  from  which  poisoning  had  occurred. 
Rorsch  and  Fassbender  state  that  while  in  some  of  its 
reactions  this  substance  resembles  digitaline,  it  is  distin- 
guished from  this  vegetable  alkaloid  by  the  failure  of  the 
ptomaine  to  give  the  characteristic  bitter  taste. 

Schwanert,  whilst  examining  the  decomposing  intes- 
tines, liver,  and  spleen  of  a  child  which  had  died  suddenly, 
perceived  a  peculiar  odor  and  obtained  by  the  Stas-Otto 
method  (ether  extract  from  an  alkaline  solution)  small 
quantities  of  a  base,  which  was  distinguished  from  nicotine 


HISTOKICAL    SKETCH.  29 

and  coniine  by  its  greater  volatility  and  its  peculiar  odor. 
He  supposed  that  this  substance  was  produced  by  decom- 
position, and,  in  order  to  ascertain  the  truth  of  his  suppo- 
sition, he  took  the  organs  of  a  cadaver  that  had  lain  for 
sixteen  days  at  a  temperature  of  30°  and  was  well  decom- 
jwsed.  These  were  treated  with  tartaric  acid  and  alcohol. 
The  acid  solution  was  first  extracted  with  ether,  and  yielded 
no  result ;  it  was  then  rendered  alkaline  and  extracted 
with  ether.  The  latter  extract  gave,  on  evaporation,  the 
same  substance  which  he  had  found  in  the  organs  of  the 
child.  The  residue  was  a  yellowish  oil,  having  an  odor 
somewhat  similar  to  propylamine.  It  was  repulsive,  but 
not  bitter  to  the  taste,  and  alkaline  in  reaction.  On  the 
addition  of  hydrochloric  acid,  it  crystallized  in  white  needles, 
which  were  freely  soluble  in  water,  but  soluble  with  diffi- 
culty in  alcohol.  On  the  addition  of  ammonium  hydrate 
to  this  crystalline  substance,  a  white  vapor  of  unpleasant 
odor  was  given  off.  The  crystals  dissolved  in  sulphuric  acid, 
forming  a  solution  which  was  at  first  colorless,  but  which 
gradually  became  dirty  brownish -yellow,  and  grayish- 
brown  on  the  application  of  heat.  On  being  warmed  with 
sodium  molybdate,  a  splendid  blue  color,  becoming  gradu- 
ally gray,  was  produced.  Potassium  bichromate  and  sul- 
phuric acid  gave  a  reddish-brown,  then  a  grass-green  color. 
Nitric  acid  gave  a  yellow  color.  A  tartaric  acid  solution 
of  the  crystals  produced,  on  the  addition  of  platinum  chlo- 
ride, a  dirty  yellow  precipitate  of  small  six-sided  stars, 
which  contained  31.55  per  cent,  of  platinum.  Gold  chlo- 
ride gave  a  pale  yellow,  amorphous  precipitate;  mercuric 
chloride  yielded  white  crystals  ;  potassio-mercuric  iodide  a 
dirty-white  precipitate  ;  and  potassio-cadmic  iodide  yielded 
no  result.  Tannic  acid  produced  only  a  turbidity.  Sodium 
phospho-molybdate  gave  a  yellow,  flocculent  precipitate, 
which  became  blue  on  the  addition  of  ammonium  hydrate. 
This  base  has  a  slight  reducing  power,  and  in  this  it 
resembles  a  substance  obtained  by  Selmi,  but  it  differs 
from  Selmi's  extract  inasmuch  as  it  does  not  give  a  violet 
coloration  on  being  warmed  with  sulphuric  acid.  In  its 
amorphous  character,  its  behavior  to  the  general  alkaloidal 


30  BACTEEIAL    POISONS. 

reagents,  and  its  lack  of  bitter  taste,  it  resembles  the  base 
obtained  by  Robsch  and  Fassbendee,  but,  unlike  that 
alkaloid,  it  is  extractable  from  alkaline  solutions  only. 

Selmi,  in  commenting  upon  the  base  studied  by  Roesch 
and  Fassbendee,  Schwanebt,  and  himself,  believing 
that  all  were  dealing  with  the  same  body,  states  that  it 
does  not  contain  phosphorus,  and  that  it  is  separated  with 
extreme  difficulty  from  the  vegetable  alkaloids. 

Liebeemann,  in  examining  the  somewhat  decomposed 
stomach  and  intestines  in  a  case  of  suspected  poisoning, 
found  an  alkaloid  al  body  which  was  unlike  that  studied  by 
the  chemists  mentioned  above,  inasmuch  as  it  was  not  vola- 
tile. The  Stas-Otto  method  was  employed.  The  ether 
extract  from  alkaline  solution  left,  on  evaporation,  a  brown- 
ish, resinous  mass,  which  dissolved  in  water  to  a  turbid 
solution,  the  cloudiness  increasing  on  heating.  This  reac- 
tion  agrees  with  coniine,  but  the  odor  differed  from  that  of 
the  vegetable  alkaloid.  The  aqueous,  strongly  alkaline 
solution  gave  the  following  reactions  : 

(1)  With  tannic  acid,  a  white  precipitate. 

(2)  With  potassium  iodide,  a  yellowish-brown,  turning 
to  dark-brown  precipitate. 

(3)  With  chlorine  water,  a  marked  white  cloudiness. 

(4)  With  phospho-molybdic  acid,  a  yellow  precipitate. 

(5)  With  potassio-mercuric  iodide,  a  white  precipitate. 

(6)  With  mercuric  chloride,  a  white  cloudiness. 

(7)  With  concentrated  sulphuric  acid,  after  a  while,  a 
reddish-violet  coloration. 

(8)  With  concentrated  nitric  acid,  after  evaporation,  a 
yellowish  spot. 

These  reactions  exclude  all  vegetable  alkaloids  save 
coniine.  The  putrefactive  alkaloid  does  not  distil  when 
heated  on  the  oil-bath  to  200°,  while  coniine  distils  at 
135°.  The  former  is  with  certainty  distinguished  from 
coniine  by  its  non-poisonous  properties. 

This  substance  is  extracted  by  ether  from  acid,  as  well 
as  from  alkaline  solutions.  The  yellow,  oily  drops  ob- 
tained after  the  evaporation  of  the  ether  are  soluble  in 
alcohol.     The  taste  is  slightly  burning. 


HISTORICAL    SKETCH.  31 

Selmi  obtained  from  both  putrefying  and  fresh  intes- 
tines a  substance  which  gave  the  general  alkaloidal  reac- 
tions with  potassium  iodide,  gold  chloride,  platinum  chlo- 
ride, potassio-mercuric  iodide,  and  phospho-molybdic  acicl. 
It  has  strong  reducing  power,  and  when  warmed  with 
sulphuric  acid  gives  a  violet  coloration.  These  reactions 
are  not  due  to  leucine,  tyrosine,  creatine,  or  creatinine. 
This  is  the  substance  which,  as  has  been  stated,  Selmi  con- 
sidered identical  with  that  observed  by  Rorsch  and  Fass- 
bender  and  Schwanert.  The  minor  differences  observed 
by  the  different  chemists  may  have  been  due  to  the  varying 
degrees  of  purity  in  which  the  substance  was  obtained  by 
them. 

From  human  bodies  which  had  been  dead  from  one  to 
ten  months,  Selmi  removed  many  alkaline  bases.  From 
an  ether  solution  of  a  number  of  these,  one  was  removed 
by  treatment  with  carbonic  acid  gas.  One  base  which  was 
insoluble  in  ether,  but  readily  soluble  in  amylic  alcohol, 
was  found  to  be  a  violent  poison,  producing  in  rabbits 
tetanus,  marked  dilatation  of  the  pupils,  paralysis,  and 
death. 

Parts  of  a  human  body  preserved  in  alcohol  were  found 
by  Selmi  to  yield  an  easily  volatile,  phosphorus-containing 
substance,  which  is  soluble  in  ether  and  carbon  disulphide, 
and  gives  a  brown  precipitate  with  silver  nitrate.  It  is 
not  the  phosphide  of  hydrogen.  A  similar  substance  is 
produced  by  the  slow  decomposition  of  the  yolks  of  eggs. 
With  potassium  hydrate  it  gives  off  ammonia  and  yields  a 
substance  having  an  intense  coniine  odor.  It  is  volatile 
and  reduces  phosphomolybdic  acid. 

Selmi  also  obtained  from  decomposing  egg-albumin  a 
body,  whose  chloride  forms  in  needles,  and  which  has  a 
curare-like  action  on  frogs.  From  one  arsenical  body  which 
had  been  buried  for  fourteen  days,  he  obtained,  by  extract- 
ing from  an  alkaline  (made  alkaline  with  baryta)  solution 
with  ether,  a  substance  which  formed  in  needles  and  which 
gave  crystalline  salts  with  acids.  With  sulphuric  acid  it 
gave  a  red  color ;  with  iodic  acid  and  sulphuric  acid  it 
liberated  free  iodine  and  gave  a  violet  coloration ;   with 


32  BACTEEIAL    POISONS. 

nitric  acid  it  gave  a  beautiful  yellow,  which  deepened  on 
the  addition  of  caustic  potash.  Platinum  chloride  gave  no 
precipitate  save  in  highly  concentrated  solutions.  From  a 
second  arsenical  body,  Selmi  obtained  by  the  same  method 
a  substance  which  gave,  with  tannic  acid,  a  white  precipi- 
tate •  with  iodine  in  hydriodic  acid  a  kermes-brown ;  with 
gold  chloride  a  yellow,  which  was  soon  reduced  ;  with 
mercuric  chloride  a  white;  with  picric  acid,  a  yellow, 
which  gradually  formed  in  crystalline  tablets.  This  sub- 
stance did  not  contain  any  arsenic,  but  was  highly  poi- 
sonous. From  the  stomach  of  a  hog,  which  had  been  pre- 
served in  a  solution  of  arsenious  acid,  Selmi  separated  an 
arsenical  organic  base.  The  fluid  was  distilled  in  a  current 
of  hydrogen.  The  distillate,  which  was  found  to  be  strongly 
alkaline,  was  neutralized  with  hydrochloric  acid  and  evapo- 
rated to  dryness,  when  cross-shaped  crystals,  giving  an  odor 
similar  to  that  of  trimethylamine,  were  obtained.  This  sub- 
stance was  found  by  Ciaccta  to  be  highly  poisonous,  pro- 
ducing strychnia-like  symptoms.  With  iodine  in  hydriodic 
acid  it  is  said  to  give  a  gray,  crystalline  precipitate. 

From  the  liquid  which  remained  in  the  retort,  a  non- 
volatile arsenical  ptomaine  was  extracted  with  ether.  An 
aqueous  solution  of  this  gave  with  tannic  acid  a  slowly 
forming,  yellowish  precipitate,  and  similarly  colored  pre- 
cipitates with  iodine  in  hydriodic  acid,  platinum  chloride, 
auric  chloride,  mercuric  chloride,  potassio-mercuric  iodide, 
potassio-bismuthic  iodide,  picric  acid,  and  potassium  bi- 
chromate. The  physiological  action  of  this  substance  as 
demonstrated  on  frogs  was  unlike  that  of  the  arsines,  but 
consisted  of  torpor  and  paralysis. 

Moriggia  and  Battistini  experimented  with  alkaloids, 
obtained  from  decomposing  bodies,  upon  guiaea-pigs  and 
frogs,  but  did  not  attempt  their  isolation  because  of  the 
rapid  decomposition  which  they  undergo  when  exposed  to 
the  air  and  by  which  they  lose  their  poisonous  properties. 
These  alkaloids  they  found  to  be  easily  soluble  in  amy  lie 
alcohol,  less  soluble  in  ether. 

In  1871  Lombroso  showed  that  the  extract  from  mouldy 
corn-meal  produced  tetanic  convulsions  in  animals.     This 


HISTORICAL    SKETCH.  33 

threw  some  light  upon  the  cases  of  sporadic  illness  which 
had  long  been  known  to  occur  among  the  peasants  of  Lom- 
bardy,  who  eat  fermented  and  mouldy  corn-meal.  In  1876 
Brugnatelli  and  Zenoni  obtained  by  the  Stas-Otto 
method  from  this  mouldy  meal  an  alkaloidal  substance 
which  was  white,  non-crystalline,  unstable,  and  insoluble 
in  water,  but  readily  soluble  in  alcohol  and  ether.  With 
sulphuric  acid  and  bichromate  of  potassium  it  yields  a 
color  reaction  very  similar  to  that  of  strychnine. 

The  action  of  the  ether  extracts  from  decomposed  brain 
resembles  that  of  curare,  but  is  less  marked  and  more 
transitory.  The  beats  of  the  frog's  heart  were  decreased  in 
number  and  strengthened  in  force ;  the  nerves  and  the 
muscles  lost  their  irritability,  and  the  animal  passed  into 
a  condition  of  complete  torpor.  The  pupils  were  dilated. 
Guareschi  and  Mosso,  using  the  Stas-Otto  method, 
obtained  from  human  brains  which  had  been  allowed  to 
decompose  at  a  temperature  of  from  10°  to  15°  for  from 
one  to  two  months,  both  volatile  and  non-volatile  bases. 
Among  the  former  only  ammonia  and  trimethylamine  were 
in  sufficient  quantity  for  identification.  With  these,  how- 
ever, were  minute  traces  of  ptomaines. 

They  obtained  non-volatile  bases  from  both  acid  and 
alkaline  solutions.  From  the  former  they  separated  a  sub- 
stance which  gave  precipitates  with  gold  chloride,  phospho- 
tungstic  acid,  phospho-molybdic  acid,  Mayer's  reagent, 
palladium  chloride,  picric  acid,  iodine  in  potassium  iodide, 
and  slightly  with  tannic  acid.  This  substance  was  not 
precipitated  with  platinum  or  mercury. 

From  the  alkaline  extract  there  was  obtained  a  substance 
which  in  dilute  hydrochloric  acid  solutions  gave  with  gold 
chloride  a  heavy  yellow  precipitate  with  reduction,  also 
precipitates  with  phospho-molybdic  acid,  platinum  chloride, 
Mayer's  reagent,  picric  acid,  phospho-tungstic  acid, 
Marme's  reagent,  iodine  in  potassium  iodide,  tannin,  bi- 
chromate of  potassium,  palladium  chloride,  and  mercuric 
chloride.  It  reduces  ferric  salts.  From  decomposed  fibrin 
the  same  investigators  obtained  one  well-defined  ptomaine. 
Analyses  of  the  platinum  compound  of  this  substance  gave 


34  BACTEEIAL    POISONS. 

the  formula  C10H15N.  This  substance  will  be  discussed  in 
a  future  chapter. 

From  fresh  brain  substance  they  separated  ammonia, 
trimethylainine,  and  an  undetermined  base.  These,  how- 
ever, are  not  to  be  regarded  as  products  of  putrefaction, 
but  as  resulting  from  the  action  of  the  reagents  upon  the 
brain  substance.  The  trimethylamine  probably  arises  from 
the  splitting  up  of  lecithin,  while  the  undetermined  base 
is  most  likely  choline,  which  also  results  from  the  breaking 
up  of  the  lecithin  molecule. 

They  also  show  that  when  Dragendorff's  method  is 
used  basic  substances  can  be  obtained  from  fresh  meat,  and 
these  are  shown  to  be  produced  by  the  action  of  the  sul- 
phuric acid  on  the  flesh. 

To  Brieger,  of  Berlin,  is  due  the  credit  of  isolating 
and  determining  the  composition  of  a  number  of  ptomaines. 
From  putrid  flesh  he  obtained  neuridine,  C5H14lSr2,  and 
neurine,  C5H13NO.  The  former  is  inert,  while  the  latter  is 
poisonous.  From  decomposed  fish  he  separated  a  poisonous 
base,  C2H4  (NH2)2,  which  is  an  isomeride  of  ethylenediamine, 
muscarine,  C5H15N03,  and  an  inert  substance,  C7H17N02, 
gad i nine.  Rotten  cheese  yielded  neuridine  and  trimethyla- 
mine. Decomposed  glue  gave  neuridine,  dimethylamine, 
and  a  musearine-like  base.  In  the  cadaver,  he  has  found 
in  different  stages  of  decomposition,  choline,  neuridine,  tri- 
methylamine, cadaverine,  C5H14N2,  putrescine,  C4H12N2,  and 
saprine,  C5H16N2.  These  are  all  inert.  After  fourteen 
days  of  decomposition  he  found  a  poisonous  substance, 
mydaleine.  From  a  cadaver  which  had  been  kept  at 
from  —  9°  to  +  5°  for  four  months,  Brieger  obtained 
mydine,  C8HuNO,  the  poisonous  substance  mydatoxine, 
C6H13]Sr02,  also  the  poison  methyl-guanidine.  From 
poisonous  mussel  he  separated  mytilotoxine,  C6H15N02. 
From  pure  cultures  of  the  typhoid  bacillus  of  Koch  and 
Eberth,  Brieger  obtained  a  poison,  typhotoxine,  and, 
from  like  cultures  of  the  tetanus  germ  of  Rosenbach, 
tetanine.  All  of  these  bases  will  be  discussed  in  detail  in 
a  subsequent  chapter. 


HISTOEICAL    SKETCH.  35 

Gautier  and  Etard  have  also  isolated  ptomaines  which 
will  be  described  later. 

In  1885,  Vaughan  succeeded  in  isolating  an  active 
agent  from  poisonous  cheese,  to  which  he  gave  the  name 
tyrotoxicon.  This  discovery  has  been  confirmed  by  New- 
ton, Wallace,  Schaffer,  Stanton,  Firth,  Ladd, 
Wolff,  Kimura,  Davis,  and  Kinnicutt. 

Nicati  and  Rietsch,  Koch,  and  others,  have  shown 
the  presence  of  a  poisonous  substance  in  cultures  of  the 
cholera  bacillus.  Salmon  and  Smith  have  done  the  same 
with  cultures  of  the  swine-plague  germ  ;  Hoffa,  with  those 
of  the  anthrax  bacillus ;  and  Brieger  with  those  of  the 
tetanus  germ. 

In  1888,  Christmas  obtained  from  cultures  of  the 
staphylococcus  pyogenes  aureus  a  proteid  which,  when  in- 
jected into  the  anterior  chamber  of  the  eye  or  under  the 
skin,  causes  suppuration. 

In  1889,  Hankin  isolated  from  cultures  of  the  bacillus 
anthracis  a  poisonous  albumose,  which,  when  employed  in 
large  doses,  proves  fatal,  and  in  small  doses  gives  immunity. 

In  1888,  Roux  and  Yersin  showed  that  the  chemical 
poison  of  Loffler's  diphtheria  bacillus  is  a  proteid  body 
which  they  believed  to  be  of  the  nature  of  a  ferment.  In 
1890,  this  work  was  continued  by  Brieger  and  Frankel 
in  their  memorable  contribution  on  bacterial  poisons,  in 
which  they  detail  the  methods  by  which  they  isolate  their 
"  toxalbumins "  from  cultures  of  the  Loffler  bacillus, 
the  anthrax  bacillus,  Eberth's  germ,  the  cholera  vibrio, 
and  the  staphylococcus  pyogenes  aureus.  Martin  made  a 
more  detailed  study  of  the  albumoses  of  anthrax.  Vaughan 
reported  poisonous  proteids  in  cultures  of  two  toxicogenic 
germs  found  in  drinking-water,  also  in  cultures  of  three  of 
Booker's  summer  diarrhoea  germs  and  in  poisonous  cheese. 
Now  and  Schweinitz  found  both  basic  and  proteid 
poisons  in  cultures  of  the  swine-plague  bacillus. 

Many  other  contributions  have  been  made,  many  of 
which  will  be  mentioned  in  subsequent  chapters. 


CHAPTER  III. 

FOODS   CONTAINING   BACTERIAL   POISONS. 

Poisonous  Mussels.— Judging  from  the  symptoms 
produced,  there  seem  to  be  three  different  kinds  of  poison- 
ous mussel.  '  In  one  class,  the  symptoms  resemble  those  of 
a  true  gastro-intestinal  irritant.  Fodere  reports  the  case  of 
a  sailor,  who,  after  eating  a  large  dish  of  mussels,  suffered 
from  nausea,  vomiting,  pain  in  the  stomach,  tenesmus,  and 
rapid  pulse.  After  death,  which  occurred  within  two  days, 
the  stomach  and  intestines  were  found  inflamed  and  filled 
with  a  tenacious  mucus.  Combe  and  others  also  report 
cases  of  the  choleraic  form  of  poisoning  from  mussel. 

However,  the  symptoms  which  most  frequently  manifest 
themselves  after  the  eating  of  poisonous  mussels  are  more 
purely  nervous.  A  sensation  of  heat  and  itching  appears 
usually  in  the  eyelids,  and  soon  involves  the  whole  face, 
and  perhaps  a  large  portion  of  the  body.  An  eruption, 
usually  called  nettle-rash,  though  it  may  be  papular  or 
vesicular,  covers  the  parts.  The  itching  is  most  annoyiug, 
and  may  be  accompanied  by  marked  swelling.  There 
follows  a  distressing  asthmatic  breathing,  which  is  relieved 
by  ether.  In  some  cases  reported  by  Mohrino,  dyspnoea 
preceded  the  eruption,  the  patients  became  insensible,  the 
face  livid,  and  convulsive  movements  of  the  extremities 
were  noticed.  Burrow  reports  similar  cases  with  delirium, 
convulsions,  coma,  and  death  within  three  days. 

In  a  third  class  of  cases,  there  may  be  a  kind  of  intoxi- 
cation resembling  somewhat  that  of  alcohol,  then  paralysis, 
coma,  and  death. 

In  1827,  Combe  observed  thirty  persons  poisoned,  two 
of  them  fatally,  with  mussels.  He  describes  the  symptoms 
as  follows :  "  None,  so  far  as  I  know,  complained  of  any- 
thing peculiar  in  the  smell  or  taste  of  the  animals,  and 


POISONOUS    MUSSELS.  37 

none  suffered  immediately  after  taking  them.  In  general, 
an  hour  or  two  elapsed,  sometimes  more ;  and  the  bad 
effects  consisted  rather  in  uneasy' feelings  and  debility  than 
in  any  distress  referable  to  the  stomach.  Some  children 
suffered  from  eating  only  two  or  three ;  and  it  will  be  re- 
membered that  Robertson,  a  young  and  healthy  man,  only 
took  five  or  six.  In  two  or  three  hours  they  complained 
of  a  slight  tension  at  the  stomach.  One  or  two  had  cardi- 
algia,  nausea,  and  vomiting ;  but  these  were  not  general  or 
lasting  symptoms.  They  then  complained  of  a  prickly  feel- 
ing in  their  hands,  heat  and  constriction  of  the  mouth  and 
throat;  difficulty  of  swallowing  and  speaking  freely;  numb- 
ness about  the  mouth,  gradually  extending  to  the  arms,  with 
great  debility  of  the  limbs.  The  degree  of  muscular  de- 
bility varied  a  good  deal,  but  was  an  invariable  symptom. 
In  some  it  merely  prevented  them  from  walking  firmly, 
but  in  most  of  them  it  amounted  to  perfect  inability  to 
stand.  While  in  bed  they  could  move  their  limbs  with 
tolerable  freedom,  but  on  being  raised  to  the  perpendicular 
posture  they  felt  their  limbs  sink  under  them.  Some  com- 
plained of  a  bad,  coppery  taste  in  the  mouth,  but  in  general 
this  was  in  answer  to  what  lawyers  call  a  leading  question. 
There  was  slight  pain  of  the  abdomen,  increased  on  pres- 
sure, particularly  in  the  region  of  the  bladder,  which  organ 
suffered  variously  in  its  functions.  In  some  the  secretion 
of  urine  was  suspended,  in  others  it  was  free,  but  passed 
with  pain  and  great  effort.  The  action  of  the  heart  was 
feeble ;  the  breathing  unaffected  ;  the  face  pale,  expressive 
of  much  anxiety ;  the  surface  rather  cold ;  the  mental 
faculties  unimpaired.  Unluckily,  the  two  fatal  cases  were 
not  seen  by  any  medical  person ;  and  we  are,  therefore, 
unable  to  state  minutely  the  train  of  symptoms.  We  ascer- 
tained that  the  woman,  in  whose  house  were  five  sufferers, 
went  away  as  in  a  gentle  sleep,  and  that  a  few  moments 
before  death  she  had  spoken  and  swallowed." 

The  woman  died  within  three  hours,  and  the  other  death 
was  that  of  a  watchman,  who  was  found  dead  in  his  box 
six  or  seven  hours  after  he  had  eaten  the  mussels.     Post- 


38  BACTEEIAL    POISONS. 

mortem  examination  in  these  showed  no  abnormality.    The 
stomach  contained  some  of  the  food  partially  digested. 

The  explorer  Vancouver  reports  four  cases  similar  to 
those  observed  by  Combe.  One  of  the  sailors  died  in  five 
and  a  half  hours  after  eating  the  mussels. 

In  some  recent  cases  reported  by  Schmidtmann,  as 
quoted  by  Brieger,  the  symptoms  were  as  follows  :  Some 
dock  hands  and  their  families  ate  of  cooked  blue  mussels 
which  had  been  taken  near  a  newly  built  dock.  The 
symptoms  appeared,  according  to  the  amount  eaten,  from 
soon  after  eating  to  several  hours  later.  There  was  a  sen- 
sation of  constriction  in  the  throat,  mouth,  and  lips ;  the 
teeth  were  set  on  edge,  as  though  sour  apples  had  been 
eaten.  There  was  dizziness,  no  headache ;  a  sensation  of 
flying,  and  an  intoxication  similar  to  that  produced  by 
alcohol.  The  pulse  was  hard,  rapid  (eighty  to  ninety),  no 
elevation  of  temperature,  the  pupils  dilated  and  reaction- 
less.  Speech  was  difficult,  broken,  and  jerky.  The  limbs 
felt  heavy ;  the  hands  grasped  spasmodically  at  objects  and 
missed  their  aim.  The  legs  were  no  longer  able  to  support 
the  body,  and  the  knees  knocked  together.  There  was 
nausea,  vomiting,  no  abdominal  pain,  no  diarrhoea.  The 
hands  became  numb  and  the  feet  cold.  The  sensation  of 
cold  soon  extended  over  the  entire  body,  and  in  some  the 
perspiration  flowed  freely.  There  was  a  feeling  of  suffoca- 
tion, then  a  restful  and  dreamless  sleep.  One  person  died 
in  one  and  three-quarters  of  an  hour,  another  in  three  and 
one-half  hours,  and  a  third  in  five  hours,  after  eating  of 
the  mussels. 

In  one  of  these  fatal  cases  rigor  mortis  was  marked  and 
remained  for  twenty-four  hours.  The  vessels  of  all  the 
organs  were  distended,  only  the  heart  was  empty.  ViR- 
CHOW  concluded  from  the  conditions  observed  that  the 
blood  had  absorbed  oxygen  with  great  avidity.  There  was 
marked  hyperemia  and  swelling  of  the  mucous  membrane 
of  the  stomach  and  intestines,  which  ViRCHOW  pronounced 
'  an  enteritis.  The  spleen  was  enormously  enlarged  and  the 
liver  showed  numerous  hemorrhagic  infarctions. 

Many  theories  have  been  advanced  to  account  for  poison- 


POISONOUS    MUSSELS  39 

ous  mussels.  It  was  formerly  believed  that  the  effects  were 
due  to  copper  which  the  auimals  obtaiued  from  the  bottoms 
of  vessels ;  but,  as  Christison  remarks,  copper  does  not 
produce  these  symptoms.  Moreover,  Christison  made 
analysis  of  the  mussels  which  produced  the  symptoms  ob- 
served by  Combe,  and  was  unable  to  detect  any  copper. 
Bouchard  at  found  copper  in  some  poisonous  mussels,  but 
he  does  not  state  the  amount  of  the  copper  nor  the  source 
of  the  animals. 

Edwards  advanced  the  theory  that  the  symptoms  were 
wholly  due  to  idiosyncrasy  in  the  consumer.  This  may  be 
true  in  some  instances  where  only  one  or  two  of  those  par- 
taking of  the  food  are  affected,  but  it  certainly  is  not  a 
tenable  hypothesis  in  such  instances  as  those  reported  by 
Combe  and  Schmidtmann,  where  a  large  number  or  all 
those  who  partook  of  the  food  were  affected. 

Coldstream  found  the  livers  of  the  Leith  mussels,  as 
he  thought,  larger,  darker,  and  more  brittle  than  normal, 
and  to  this  diseased  condition  he  attributed  the  ill  effects. 

Lamoroux,  Mohring,  de  Beume,  Chenu,  and  du 
Rondeau  have  supposed  that  the  poisonous  effects  were 
due  to  a  particular  species  of  medusae  upon  which  the  mus- 
sels feed.  De  Beume  found  in  the  vomited  matter  of  one 
person,  suffering  from  mussel  poisoning,  some  medusa?,  and 
he  states  that  these  are  most  abuudaut  during  the  summer, 
when  mussels  are  most  frequently  found  to  be  poisonous. 

The  theory  of  Burrow  that  the  animal  is  always  poison- 
ous during  the  period  of  reproduction  has  been  received 
with  considerable  credit.  However,  cases  of  poisoning  have 
occurred  at  different  seasons  of  the  year. 

Crumpe,  in  1872,  suggested  that  there  is  a  species  of 
mussel  which  is  in  and  of  itself  poisonous,  and  this  species 
is  often  mixed  with  the  edible  variety.  Schmidtmann  and 
Virchow  support  this  idea.  They  state  that  the  poisonous 
species  has  a  brighter  shell,  a  sweeter,  more  penetrating, 
bouillon-like  odor  than  the  edible  kind,  also  that  the  flesh 
of  the  former  is  yellow  and  that  the  water  in  which  they 
are  cooked  is  bluish.  Lohmeyer  also  champions  this 
opinion.     This  theory,  however,  is  opposed  by  the  majority 


40  BACTEEIAL    POISONS. 

of  zoologists.  Mobius  states  that  the  peculiarities  of  the 
supposed  poisonous  variety  pointed  out  by  Viechow  and 
Schmidtmann  are  really  due  to  the  conditions  under  which 
tlie  animal  lives,  the  amount  of  salt  in  the  water,  the  tem- 
perature of  the  water,  whether  it  is  moving  or  still  water, 
the  nature  of  the  bottom,  etc.  Finally,  Mobius  states  that 
the  sexual  glands,  which  form  the  greater  part  of  the 
mantle,  are  white  in  the  male  and  yellow  in  the  female. 
However,  it  has  been  shown  later  by  Schmidtmann  and 
Vibchow  that  edible  mussels  may  become  poisonous  if  left 
in  filthy  water  for  fourteen  days  or  longer,  and,  on  the 
other  hand,  poisonous  ones  may  become  fit  for  food  if  kept 
for  four  weeks  in  good  water. 

Cats  and  dogs  which  have  eaten  voluntarily  of  poisonous 
mussels  have  suffered  from  symptoms  similar  to  those  ob- 
served in  man  ;  and  rabbits  have  been  poisoned  by  the 
administration  of  the  water  in  which  the  food  has  been 
cooked.  A  rabbit  which  was  treated  in  this  manner  by 
Schmidtmann  died  within  one  minute.  From  these 
mussels  Briegee  extracted  the  ptomaine  mytilotoxine, 
which  will  be  discussed  in  a  subsequent  chapter.  This 
poison  has  a  curare-like  action.  Whether  or  not  those 
mussels  which  produce  other  symptoms  also  contain  pto- 
maines, remains  for  future  investigations  to  determine. 

In  1887  three  other  cases  of  mussel  poisoning,  one  fatal 
case,  occurred  at  Wilhelmshaven,  the  place  which  supplied 
Beieger  with  the  mussels  from  which  he  obtained  mytilo- 
toxine. Schmidtmann  has  found  that  non-poisonous 
mussels  placed  in  the  waters  of  this  bay  soon  become  poi- 
sonous, and  that  the  poisonous  mussels  from  the  bay  placed 
in  the  open  sea  soon  lose  their  poisonous  properties.  Lin- 
dee  has  found  in  the  water  of  the  bay  and  in  the  mussels 
living  in  it  a  great  variety  of  protozoa,  amoeba,  bacteria, 
and  other  lower  organisms,  which  are  not  found  in  the 
water  of  the  open  sea  nor  in  the  non-poisonous  mussel.  He 
has  also  found  that,  if  the  water  of  the  bay  be  filtered,  non- 
poisonous  mussels  in  it  do  not  become  poisonous.  He 
therefore  concludes  that  poisonous  mussels  are  those  which 
are  suffering  from  disease  due  to  residence  in  filthy  water. 


POISONOUS    FISH.  41 

Brieger  has  tested  dead  and  decomposed  mussels  taken 
from  the  open  sea  for  mytilotoxine,  with  negative  results. 

Poisonous  Oysters  and  Eels. — Pasquier  reported 
cases  of  poisoning  at  Havre  from  the  eating  of  oysters 
taken  from  an  artificial  bed  which  had  been  established 
near  the  outlet  of  a  drain  from  a  public  water-closet. 
Christison  says  that  an  "  unusual  prevalence  of  colic, 
diarrhoea,  and  cholera"  at  Dunkirk  was  believed  to  have 
been  traced  to  an  importation  of  unwholesome  oysters  from 
the  Normandy  coast.  Vaughan  and  Novy  obtained 
tests  for  tyrotoxicon  in  the  liquor  of  some  decomposed 
oysters  which  had  caused  illness  in  many  people  at  a  church 
festival. 

Virey  states  that  many  persons  were  attacked  with 
violent  pain  and  diarrhoea  a  few  hours  after  eating  a  pate 
made  of  eels  from  a  stagnant  cattle-ditch  near  Orleans, 
also  that  similar  cases  have  occurred  in  various  parts  of 
France,  and  that  domestic  animals  have  been  killed  by 
eating  the  remains  of  the  poisonous  dish. 

Poisonous  Fish. — While  many  species  of  fish  are  popu- 
larly regarded  as  poisonous,  but  little  scientific  work  has 
been  done  in  this  line,  and  we  are  not  prepared  to  say  to 
what  extent  this  popular  idea  is  correct.  Miura  and 
Takesaki  find  that  the  ripe  ovaries  of  tetrodon  rubripes 
contain  a  substance  which  induces  in  rabbits  acceleration 
of  the  respiratory  movements,  paralysis  of  the  skeletal 
muscles,  mydriasis,  increased  peristalsis  of  the  intestines, 
and  arrest  of  the  heart. 

The  disease  known  as  "  kakke,"  which  prevails  from 
May  to  October  in  Tokio  is,  according  to  Miura  and 
others,  an  intoxication  due  to  the  eating  of  fish,  which  be- 
long to  the  soomb7'idce.  The  affection  is  generally  chronic 
or  subacute,  seldom  acute.  The  most  characteristic  symp- 
tom is  paralysis  of  the  diaphragm  with  consequent  dysp- 
noea and  disturbance  of  the  action  of  the  heart.  Electri- 
cal stimulation  of  the  diaphragm  has  proven  to  be  the 
most  successful  treatment. 


42  BACTERIAL    POISONS. 

Sausage  Poisoning. — This  is  also  known  as  botulis- 
mus  and  allantiasis.  While  considerable  diversity  has 
been  observed  in  symptoms  of  sausage  poisoning,  we  can- 
not divide  the  cases  into  classes  from  their  symptoma- 
tology as  has  been  done  in  mussel  poisoning.  The  first 
effects  may  manifest  themselves  at  any  time  from  one  hour 
to  twenty-four  hours  after  eating  of  the  sausage,  and  cases 
are  recorded  in  which  it  is  stated,  no  symptoms  appeared 
until  several  days  had  passed.  However,  we  must  re- 
member that  trichinosis  was  frequently,  in  former  times, 
classed  as  sausage  poisoning,  and  it  is  highly  probable  that 
these  cases  of  long  delay  in  the  appearance  of  the  symp- 
toms were  really  not  due  to  putrefaction,  but  to  the  pres- 
ence of  parasites  in  the  meat.  A  large  majority  of  the  one 
hundred  and  twenty-four  cases  more  recently  reported  by 
Muller  sickened  within  twenty-four  hours,  and  out  of 
the  forty-eight  of  these  which  were  fatal,  six  died  within 
the  first  twenty-four  hours.  At  first  there  is  dryness  of 
the  mouth,  constriction  of  the  throat,  uneasiness  in  the 
stomach,  nausea,  vomiting,  vertigo,  indistinctness  of  vision, 
dilatation  of  the  pupils,  difficulty  in  swallowing,  and 
usually  diarrhoea,  though  obstinate  constipation  may  exist 
from*  the  first.  There  is,  as  a  rule,  a  sensation  of  suffoca- 
tion, and  the  breathing  becomes  labored.  The  pulse  is 
small,  thready,  and  rapid.  In  some  cases  the  radial  pulse 
may  be  imperceptible.  Marked  nervous  prostration  and 
muscular  debility  follow.  These  symptoms  vary  greatly 
in  prominence  in  individual  cases.  The  rechting  and  vom- 
iting, which  may  be  most  distressing  and  persistent  in 
some  instances,  in  others  are  trivial  at  the  beginning  and 
soon  cease  altogether.  The  same  is  true  of  the  diarrhoea. 
As  a  rule,  the  functions  of  the  brain  proceed  normally,  but 
there  may  be  delirium,  then  coma  and  death.  In  some 
there  are  marked  convulsive  movements,  especially  of  the 
limbs,  in  others  paralysis  may  be  an  early  and  marked 
symptom.  The  pupils  may  dilate,  then  become  normal 
and  again  dilate.  There  is  frequently  ptosis,  and  paralysis 
of  the  muscles  of  accommodation  is  not  rare.  Complete 
blindness  has  followed  in  a  few  instances. 


SAUSAGE    POISONING.  43 

The  fatality  varies  greatly  in  different  outbreaks.  In 
1820  Kerner  collected  reports  of  seventy-six  cases,  of 
which  thirty-seven  were  fatal.  In  his  next  publication 
(1822)  he  increased  the  number  to  one  hundred  and  fifty- 
five  cases,  with  eighty-four  fatal  results.  This  gave  a 
mortality  of  over  fifty  per  cent.,  while  in  one  outbreak 
reported  by  Muller  the  mortality  was  less  than  two  per 
cent. 

A  large  proportion  of  the  cases  of  sausage  poisoning 
have  occurred  in  Wiirtemberg  and  the  immediately  adja- 
cent portions  of  Baden.  This  fact  has,  without  doubt, 
been  correctly  ascribed  to  the  methods  there  practised  of 
preparing  and  curing  the  sausage.  It  is  said  to  be  com- 
mon for  the  people  to  use  the  blood  of  the  sheep,  ox,  and 
goat  in  the  preparation  of  this  article  of  diet.  Moreover, 
the  blood  is  kept  sometimes  for  days  in  wooden  boxes  and 
at  a  high  temperature  before  it  is  used.  In  these  cases  it 
is  altogether  likely  that  putrefaction  progresses  to  the  poi- 
sonous stage  before  the  process  of  curing  is  beguu.  How- 
ever, cases  of  poisoning  have  occurred  from  beef  and  pork 
sausages  as  well. 

Moreover,  the  method  of  curing  employed  in  Wurtem- 
berg favors  putrefaction.  A  kind  of  sausage  known  as 
"blunzen"  is  made  by  filling  the  stomachs  of  hogs  with 
the  meat.  In  curing,  the  interior  of  this  great  mass  is  not 
acted  upon,  and  putrefaction  sets  in.  The  curing  is  usually 
done  by  hanging  the  sausage  in  the  chimney.  At  night 
the  fire  often  goes  out  and  the  meat  freezes.  The  alternate 
freezing  and  thawing  render  decomposition  more  easy. 
The  interior  of  the  sausage  is  generally  the  most  poison- 
ous. Indeed,  in  niauy  instances  those  who  have  eaten  of 
the  outer  portion  have  been  unharmed,  while  those  who 
have  eaten  of  the  interior  of  the  same  sausage  have  been 
most  seriously  affected. 

Many  German  writers  state  that  when  a  poisonous  saus- 
age is  cut,  the  putrid  portion  has  a  dirty,  grayish-green 
color,  and  a  soft,  smeary  consistency.  A  disagreeable 
odor,  resembling  that  of  putrid  cheese,  is  perceptible.  The 
taste  is  unpleasant,  and  sometimes  a  smarting  of  the  mouth 


44  BACTERIAL    POISONS. 

and  throat  is  produced.  Post-mortem  examination  after 
sausage  poisoning  shows  no  characteristic  lesion.  It  is 
generally  stated  that  putrefaction  sets  in  very  tardily,  but 
Muller  shows  that  no  reliance  can  be  placed  upon  this 
point,  and  states  that  out  of  forty-eight  recorded  autopsies, 
it  was  especially  stated  in  eleven  that  putrefaction  rapidly 
developed.  In  some  instances  there  has  been  noticed 
hyperemia  of  the  stomach  and  intestinal  canal,  but  this  is 
by  no  means  constant.  The  liver  and  brain  have  been  re- 
ported as  congested,  but  this  would  result  from  the  failure 
of  the  heart,  and  would,  by  no  means,  be  characteristic  of 
poisoning  with  sausage. 

Yon  Faber,  in  1821,  observed  sixteen  persons  who 
were  made  sick  by  eating  fresh,  uusmoked  sausage  made 
from  the  flesh  of  a  pig  which  had  suffered  from  an  abscess 
on  the  neck.  Five  of  the  patients  died.  The  symptoms 
were  as  follows :  There  was  constriction  of  the  throat, 
difficulty  in  swallowing,  retching,  vomiting,  colic-like 
pains,  vertigo,  hoarseness,  dimness  of  vision,  and  headache. 
Later  and  in  severer  cases,  there  was  complete  exhaustion, 
and,  finally,  paralysis.  The  eyeballs  were  retracted,  the 
pupils  were  sometimes  dilated,  then  contracted ;  they  did 
not  respond  to  light ;  there  was  paralysis  of  the  upper  lids. 
The  tonsils  were  swollen,  but  not  as  in  tonsillitis.  Liquids 
which  were  not  irritating  could  be  carried  as  far  as  the 
oesophagus,  when  they  were  then  ejected  from  the  mouth 
and  nose  with  coughing.  Solid  foods  could  not  be  swal- 
lowed. On  the  back  of  the  tongue  and  in  the  pharynx 
there  was  observed  a  puriform  exudate. 

Obstinate  constipation  existed  in  all,  while  the  sphincter 
ani  was  paralyzed.  The  breathing  was  easy,  but  all  had 
a  croupous  cough.  The  skin  was  dry.  There  was  incon- 
tinence of  urine.  There  was  no  delirium  and  the  mind 
remained  clear  to  the  last. 

Post-mortem  examinations  were  held  on  four.  The 
skin  was  rough — "goose-skin."  The  abdomen  was  re- 
tracted. The  large  vessels  in  the  upper  part  of  the  stom- 
ach were  filled  with  black  blood.  The  contents  of  the 
stomach  consisted  of  a  reddish-brown,  semi-fluid  substance, 


SAUSAGE    POISOXING.  45 

which  gave  off  a  repugnant,  acid  odor.  In  one  case  the 
omeutuni  was  found  greatly  congested.  The  large  intes- 
tine was  very  pale,  and  the  right  ventricle  of  the  heart 
was  filled  with  dark  fluid  blood. 

Schuz  cites  thirteen  cases  of  poisoning  from  liver  saus- 
age in  which  the  symptoms  differed  from  the  foregoing  in 
the  following  respects : 

(1)  In  only  one  out  of  the  thirteen  was  there  constipa- 
tion; all  the  others  had  numerous  watery,  typhoid-like 
stools. 

(2)  Symptoms  involving  the  sense  of  sight  were  present 
in  only  three;  in  all  the  pupils  were  unchanged. 

(3)  The  croupous  cough  was  wholly  wauting ;  though 
in  many  there  was  complete  loss  of  voice.  Difficulty  of 
swallowing  was  complained  of  by  only  one. 

(4)  Delirium  was  marked  in  all ;  and  in  one  the  dis- 
turbance of  the  mental  faculties  was  prominent  for  several 
wreeks. 

(5)  There  were  no  deaths. 

(6)  The  time  between  eating  the  sausage  and  the  appear- 
ance of  the  symptoms  varied  from  eighteen  to  twenty-four 
hours,  and  the  duration  of  sickness  from  one  to  four 
weeks  ;  though  in  one  case  complete  recovery  did  not  occur 
until  after  two  and  one-half  months. 

The  sausages  were  not  smoked,  and  all  observed  a  garlic 
odor,  though  no  garlic  had  been  added  to  the  meat. 

Tripe  reports  sixty -four  cases.  The  symptoms  came 
on  from  three  and  one-half  to  thirty-six  hours  after  eating. 
The  stools  were  frequent,  watery,  and  of  offensive  odor. 
In  some  there  was  delirium.  One  died.  In  the  fatal  case 
the  hands  and  face  were  cold  and  swollen.  The  pulse  was 
rapid  and  wreak.  The  pupils  were  contracted,  but  re- 
sponded to  light.    The  small  intestine  was  found  inflamed. 

Hedinger  reports  the  case  of  a  man  and  a  woman  with 
the  usual  symptoms,  but  during  recovery  the  dilatation  of 
the  pupils  was  followed  by  contraction.  Birds  ate  of  this 
sausage,  and  were  not  affected. 

Roser  reports  cases  in  which  there  were  found,  after 
death,  abscesses  of  the  tonsils,  a  dark,  bluish  appearance 

3* 


46  BACTERIAL    POISONS. 

of  the  mucous  membrane  of  the  pharynx,  larynx,  and 
bronchial  tubes,  dark  redness  of  the  fundus  of  the  stom- 
ach, and  circumscribed,  gray,  red,  and  black  spots  on  the 
mucous  membrane  of  the  intestine.  The  liver  was  brittle 
and  the  spleen  enlarged. 

Many  theories  concerning  the  nature  of  the  active  prin- 
ciple of  poisonous  sausage  have  been  advanced.  It  was 
once  believed  to  consist  of  pyroligneous  acid,  which  was 
supposed  to  be  absorbed  by  the  meat  from  the  smoke  used 
in  curing  it;  but  it  was  soon  found  that  unsmoked  sausage 
might  be  poisonous  also.  Emmert  believed  that  the  active 
agent  was  hydrocyanic  acid,  and  Jager's  theory  supposed 
the  presence  of  picric  acid.  But  these  acids  are  not  found 
in  poisonous  sausage,  and,  moreover,  their  toxicological 
effects  are  wholly  unlike  those  observed  in  sausage  poison- 
ing. As  we  have  elsewhere  seen,  Kerner  believed  that 
he  had  found  the  poisonous  principle  in  a  fatty  acid.  This 
theory  was  supported  by  Dann,  Buchner,  and  Schu- 
mann. Kerner  believed  the  poison  to  consist  of  either 
caseic  or  sebacic  acid,  or  both,  while  Buchner  named  it 
acidum  botulinicum ;  but  the  acids  of  the  former  proved 
to  be  inert,  and  that  of  the  latter  to  have  no  existence. 
Schlossberger  first  suggested  that  the  poisonous  sub- 
stance is  most  probably  basic  in  character,  and  he  found 
an  odoriferous,  ammoniacal  base  which  could  not  be  found 
in  good  sausage,  and  which  did  not  correspond  to  any 
known  amides,  imides,  or  nitril  bases.  However,  this 
substance  has  not  been  obtained  by  anyone  else,  nor  has 
it  been  demonstrated  to  be  poisonous. 

Liebig,  Duflas,  Hirsch,  and  Simon  believed  in  the 
presence  of  a  poisonous  ferment.  Van  den  Corput  de- 
scribed sarcina  botuliua,  which  was  believed  to  constitute 
the  active  agent.  Muller,  Hoppe-Seyler,  and  others 
have  found  various  microorganisms,  and  Virchow,  Eicii- 
enberg,  and  others  have  examined  microscopically  the 
blood  of  persons  poisoned  with  sausage.  Recently,  Ehr- 
enberg  has  attempted  to  isolate  the  poisonous  substance 
by  employing  Brieger's  method,  but  he  obtained  only 
inert  substances. 


POISONOUS    HAM.  47 

Gaffky  and  Paak  have  made  a  thorough  study  of 
some  sausage  which  poisoned  a  large  number  of  people, 
among  whom  one,  a  strong  man,  died.  The  sausage  was 
made  of  horse-flesh  and  liver.  In  the  majority  of  the 
persons  the  symptoms  came  on  within  six  hours  and  in 
one  instance  within  half  an  hour.  Many  had  a  severe 
chill ;  some  did  not.  The  most  prominent  symptoms  were 
headache,  loss  of  appetite,  pain  in  the  bowels,  vomiting 
and  purging.  In  the  fatal  case,  however,  there  was  no 
vomiting.  From  the  sausage  Gaffky  and  Paak  isolated 
a  short  bacillus,  which  when  given  by  the  mouth,  snb- 
cutaneously  or  intravenously  produced  the  above  symptoms, 
with  a  fatal  termination  in  most  instances,  in  rabbits, 
guinea-pigs,  mice,  and  apes.  Gaffky  and  Paak  were 
unable  to  isolate  the  chemical  poison. 

Poisonous  Ham. — Under  this  head  we  shall  not  discuss 
cases  of  poisoning  from  trichina  or  other  parasites,  but  shall 
refer  only  to  those  instances  in  which  the  toxic  agent  has 
originated  in  putrefactive  changes.  A  number  of  such 
cases  have  been  observed  within  the  past  ten  years,  but 
only  a  few  of  them  have  been  investigated  scientifically. 
The  best  known  of  these,  as  well  as  the  most  thoroughly 
studied,  is  the  Wellbeck  poisoning,  which  Ballard  in- 
vestigated successfully.  Iu  June,  1880,  a  large  number  of 
persons  attended  a  sale  of  timber  and  machinery  on  the 
estate  of  the  Duke  of  Portland  at  Wellbeck.  The  sale 
continued  four  days,  and  lunches  were  served  by  the  pro- 
prietress of  a  neighboring  hotel.  The  refreshments  con- 
sisted of  cold  boiled  ham,  cold,  boiled,  or  roasted  beef, 
cold  beefsteak  pie,  mustard  and  salt,  bread  and  cheese, 
pickles  and  Chutney  sauce.  The  drinks  were  bottle  and 
draught  beer,  spirits,  ginger  beer,  lemonade,  and  water. 
Many  were  poisoned,  and  Ballard  obtained  the  particu- 
lars of  seventy-two  cases,  among  which  there  were  four 
deaths.     The  symptoms  are  given  by  Ballard  as  follows: 

"  I  propose  to  speak  of  the  attacks  under  the  name  of 
'  diarrhoeal  illness/  because  diarrhoea  was  the  most  constant 
of  all  the  symptoms  observed,  and  the  other  symptoms 


48  BACTEKIAL    POISONS. 

were  in  some  respects  so  peculiar  that  I  am  indisposed  to 
give  to  the  disease  any  name  otherwise  generally  recognized. 
As  might  have  been  anticipated  from  our  experience  of 
diseases  in  general,  there  were  varieties  in  severity  among 
the  cases  investigated  ;  and  symptoms  strongly  marked  in 
some,  were  slightly  marked  or  altogether  wanting  in  others. 
Perhaps  I  shall  do  the  best  service  by  giving  first  a  general 
sketch  of  the  course  of  the  illness,  subsequently  illustrating 
it  by  a  description  of  a  few  well-marked  cases. 

"A  period  of  incubation  preceded  the  illness.  In  fifty- 
one  cases  where  this  could  be  accurately  determined,  it 
was  twelve  hours  or  less  in  five  cases ;  between  twelve  and 
thirty-six  hours  in  thirty-four  cases ;  between  thirty-six 
and  forty-eight  hours  in  eight  cases ;  and  later  than  this  in 
only  four  cases.  In  many  cases  the  first  definite  symptoms 
occurred  suddenly,  and  evidently  unexpectedly,  but  in  some 
cases  there  were  observed  during  the  incubation  more  or 
less  feeling  of  languor  and  ill  health,  loss  of  appetite, 
nausea,  or  fugitive,  griping  pains  in  the  belly.  In  about 
a  third  of  the  cases  the  first  definite  symptom  was  a  sense 
of  chilliness,  usually  with  rigors,  of  trembling,  in  one  case 
accompanied  by  dyspnoea;  in  a  few  cases  it  was  giddiness 
with  faintness,  sometimes  accompanied  by  a  cold  sweat  and 
tottering;  in  others,  the  first  symptom  was  headache  or 
pain  somewhere  in  the  trunk  of  the  body,  e.  g.,  in  the 
chest,  back,  between  the  shoulders,  or  in  the  abdomen,  to 
which  part  the  pain,  wherever  it  might  have  commenced, 
subsequently  extended.  In  one  case  the  first  symptom 
noticed  was  a  difficulty  in  swallowing.  In  two  cases  it  was 
intense  thirst.  But  however  the  attack  may  have  com- 
menced, it  was  usually  not  long  before  pain  in  the  abdomen, 
diarrhoea,  and  vomiting  came  on,  diarrhoea  being  of  more 
certain  occurrence  than  vomiting.  The  pain  in  several 
cases  commenced  in  the  chest  or  between  the  shoulders,  and 
extended  first  to  the  upper  and  then  to  the  lower  part  of 
the  abdomen.  It  was  usually  very  severe  indeed,  quickly 
producing  prostration  or  faintness,  with  cold  sweats.  It 
was  variously  described  as  crampy,  burning,  tearing,  etc. 
The  diarrhoeal  discharges  were  in  some  cases  quite  unre- 


POISONOUS    HAM.  49 

strai nable,  and  (where  a  description  of  them  could  be  ob- 
tained) were  said  to  have  been  exceedingly  offensive  and 
usually  of  a  dark  color.  Muscular  weakness  was  an  early 
and  very  remarkable  symptom  in  nearly  all  the  cases,  and 
in  many  it  was  so  great  that  the  patient  could  only  stand 
by  holding  on  to  something.  Headache,  sometimes  severe, 
was  a  common  and  early  symptom  ;  and  in  most  cases  there 
was  thirst,  often  intense  and  most  distressing.  The  tougue, 
when  observed,  was  described  usually  as  thickly  coated 
with  a  brown,  velvety  fur,  but  red  at  the  tip  and  edges. 
In  the  early  stage  the  skin  was  often  cold  to  the  touch,  but 
afterward  fever  set  in,  the  temperature  rising  in  some 
cases  to  101°,  103°,  and  104°  F.  In  a  few  severe  cases 
where  the  skin  was  actually  cold,  the  patient  complained  of 
heat,  insisted  on  throwing  off  the  bedclothes,  and  was  very 
restless.  The  pulse  in  the  height  of  the  illness  became 
quick,  counting  in  some  cases  100  to  128.  The  above 
were  the  symptoms  most  frequently  noted.  Other  symp- 
toms occurred,  however,  some  in  a  few  cases,  and  some  only 
in  solitary  cases.  These  I  now  proceed  to  enumerate. 
Excessive  sweating,  cramps  in  the  legs,  or  in  both  legs  and 
arms,  convulsive  flexion  of  the  hands  or  fingers,  muscular 
twitchiugs  of  the  face,  shoulders,  or  hands,  aching  pain  in 
the  shoulders,  joints,  or  extremities,  a  sense  of  stiffness  of 
the  joints,  prickling  or  tingling  or  numbness  of  the  hands 
lasting  far  into  convalescence  in  some  cases,  a  sense  of 
general  compression  of  the  skin,  drowsiness,  hallucinations, 
imperfection  of  vision,  and  intolerance  of  light.  In  three 
cases  (one,  that  of  a  medical  man)  there  was  observed  yel- 
lowness of  the  skin,  either  general  or  confined  to  the  face 
and  eyes.  In  one  case,  at  a  late  stage  of  the  illness,  there 
was  some  pulmonary  congestion,  and  an  attack  of  what  was 
regarded  as  gout.  In  the  fatal  cases,  death  was  preceded 
by  collapse  like  that  of  cholera,  coldness  of  the  surface, 
pinched,  features  and  blueness  of  the  fingers  and  toes  and 
around  the  sunken  eyes.  The  debility  of  convalescence 
was  in  nearly  all  cases  protracted  to  several  weeks. 

"The  mildest  cases  were  characterized  usually  by  little 
remarkable  beyond  the  following  symptoms,  viz.,  abdominal 


50  BACTERIAL    POISONS. 

pains,  vomiting,  diarrhoea,  thirst,  headache,  and  muscular 
weakness;  any  one  or  two  of  which  might  be  absent." 

The  cause  of  this  illness  was  traced  conclusively  to  the 
hams  eaten.  Klein  found  in  the  meat  a  bacillus,  cultures 
of  which  were  used  for  inoculating  animals.  These  inocu- 
lations were  found  generally  to  be  followed  by  pneumonia. 
No  attempt  was  made  to  isolate  a  ptomaine. 

Later,  Ballard  reported  fifteen  cases  with  symptoms 
similar  to  the  above,  aud  with  one  death,  from  eating  baked 
pork.  Not  all  of  those  who  ate  of  this  pork  were  made 
sick.  This  might  have  been  due  to  inequality  in  the  putre- 
factive changes  in  different  portions  of  the  meat,  or  it  may 
have  been  due  to  differences  in  temperature  in  various  por- 
tions of  the  meat  during  the  cooking.  In  the  blood,  peri- 
cardial fluid,  and  lungs  of  the  fatal  case,  Klein  observed 
bacilli  similar  to  those  discovered  in  the  Wellbeck  inquiry. 
Pneumonia  was  produced  by  inoculating  guinea-pigs  and 
mice  with  these  bacilli. 

In  meat  which  poisoned  a  large  number  of  persons, 
Gartner  found  his  bacillus  enteritidis.  The  meat  was 
from  a  cow  which  had  a  severe  diarrhoea  for  two  days  be- 
fore she  was  killed.  Of  twelve  persons  who  ate  the  flesh 
raw,  all  were  sick ;  while  of  those  who  ate  of  the  cooked 
food  a  large  per  cent,  were  also  affected.  In  the  meat  and 
in  the  spleen  of  a  person  who  died  from  the  effects  of  the 
poison,  Gartner  found  the  bacillus,  which  proved  fatal 
to  animals.  Good  beef,  inoculated  with  this  bacillus  and 
cooked  some  hours  later,  killed  rabbits,  guinea-pigs,  and 
mice.  The  skin  of  the  people  who  were  poisoned  and  re- 
covered peeled  off.  The  period  of  incubation  varied  from 
two  to  thirty  hours. 

August  29,  1887,  256  soldiers  and  36  citizens  at  Middle- 
burg,  Holland,  were  taken  sick  after  eating  meat  from  a 
cow  which  had  been  killed  while  suffering  from  puerperal 
fever.  The  symptoms  were  nausea,  vomiting,  purging, 
elevation  of  temperature,  and  prostration.  In  some  there 
were  observed  dizziness,  sleepiness,  aud  dilatation  of  the 
pupil.  After  a  few  days  these  symptoms  gradually  disap- 
peared, and  in  many  an  eczematous  eruption  of  the  lips 


POISONOUS    MEAT.  51 

gave  annoyance.  Pigs,  cats,  and  dogs  which  ate  of  the 
offal  of  this  animal  were  also  made  sick.  Thorough 
cooking  did  not  destroy  the  poison,  and  those  who  took 
soup  and  boullion  made  from  the  meat  were  affected  like 
those  who  ate  of  the  muscular  fibre.  In  most  of  the  cases 
the  symptoms  came  on  within  twelve  hours  after  eating 
the  meat. 

On  a  fete-day  at  Zurich,  in  1839,  600  persons  who  were 
fed  upon  cold  veal  and  ham  were  taken  ill,  with  shivering, 
giddiness,  vomiting,  and  diarrhoea.  Some  were  delirious 
and  others  were  salivated,  the  saliva  being  extremely  fetid. 
In  the  worst  cases  there  were  involuntary  stools,  collapse, 
and  death.  The  cause  was  traced  to  putrefactive  changes 
in  the  meat. 

Siedler  reports  an  instance  of  four  persons  having  been 
made  sick  by  eating  decomposed  goose-grease.  There  were 
giddiness,  prostration,  and  violent  vomiting.  No  metallic 
poison  could  be  found.  The  grease  was  rancid,  of  repul- 
sive odor,  and  three  ounces  of  it  given  to  a  dog  produced 
the  same  symptoms  which  had  been  observed  in  the 
persons. 

Christison  reports  a  number  of  cases  in  which  persons 
were  seriously,  a  few  fatally,  affected  by  eating  various 
kinds  of  meat  which  had  undergone  partial  putrefaction. 

Ollivier  found  six  persons  poisoned,  four  of  them 
fatally,  by  eating  of  decomposed  mutton.  He  also  men- 
tions the  poisoning  of  a  family  of  three  with  ham  pie. 
Chemical  analysis  failed  to  reveal  the  presence  of  any 
poison. 

Boutigny,  having  failed  to  find  any  poison  in  the  meat 
furnished  at  a  festival,  and  to  which  the  serious  illness  of 
many  was  attributed,  made  a  meal  of  stuffed  turkey  fur- 
nished by  the  same  dealer,  but  after  a  short  time  his  coun- 
tenance became  livid,  his  pulse  small  and  feeble,  a  cold 
sweat  bathed  his  body,  and  violent  vomiting  and  purging 
followed.     His  recovery  was  slow. 

Geiseler  observed  nausea,  vomiting,  purging,  and 
delirium  after  eating  of  bacon  which  was  imperfectly  cured. 


52  BACTERIAL    POISONS. 

Poisonous  Canned  Meats. — Cases  of  poisoning  from 
eating  canned  meats  have  become  quite  frequent.  Although 
it  may  be  possible  that  in  some  instances  the  untoward 
effects  result  from  metallic  poisoning,  in  the  great  majority 
of  cases  the  poisonous  principles  are  formed  by  putrefactive 
changes.  In  many  instances  it  is  probable  that  decomposi- 
tion begins  after  the  can  is  opened  by  the  consumer.  In 
others,  the  canning  is  carelessly  done  and  putrefaction  is 
far  advanced  before  the  food  reaches  the  consumer.  In 
still  other  instances,  the  meat  may  be  taken  from  diseased 
animals,  or  it  may  undergo  putrefactive  changes  before 
the  cauuing.  What  is  true  of  canned  meats  is  also  true  of 
canned  fruits  and  vegetables. 

Dr.  Ashworth,  of  Smithland,  Iowa,  has  reported  to  us 
three  fatal  cases  of  poisoning  from  canned  apricots.  An 
infant,  which  was  only  eight  days  old,  and  which  must 
have  received  the  poison  from  its  mother's  breasts,  died 
within  a  few  hours.  The  mother  died  forty-three  hours 
after  eating  the  apricots,  and  the  father  on  the  sixth  day. 
The  symptoms  corresponded  with  those  of  poisoning  by 
tyrotoxicon.  However,  it  seems  that  no  analysis  was 
made,  and  these  may  have  been  cases  of  mineral  poisoning. 

Poisonous  Cheese. — In  1827  Hunnefeld  made  some 
analyses  of  poisonous  cheese,  and  experimented  with  ex- 
tracts upon  the  lower  animals.  He  accepted  the  ideas  of 
Keener  in  regard  to  poisonous  sausage  in  a  somewhat 
modified  form,  and  thought  the  active  agents  to  be  sebacic 
and  caseic  acids.  About  the  same  time,  Serturner, 
making  analyses  of  poisonous  cheese  for  Westrumb,  also 
traced  the  poisonous  principles,  as  he  supposed,  to  these 
fatty  acids.  We  see  from  this  that  during  the  first  part  of 
the  present  century  the  fatty  acid  theory,  as  it  may  be 
called,  was  generally  accepted. 

.  In  1848,  Christison,  after  referring  to  the  work  of 
Hunnefeld  and  Serturner,  made  the  following  state- 
ment :  "  His  (Hiinnefeld's)  experiments,  however,  are  not 
quite  conclusive  of  the  fact  that  these  fatty  acids  are  really 
the  poisonous  principles,  as  he  has  not  extended  his  experi- 


POISONOUS    CHEESE  53 

mental  researches  to  the  easeic  and  sebacic  acids  prepared 
in  the  ordinary  way.  His  views  will  probably  be  altered 
and  simplified  if  future  experiments  should  confirm  the 
late  inquiries  of  Braoonnot,  who  has  stated  that  Proust's 
easeic  acid  is  a  modification  of  acetic  acid  combined  with 
an  acrid  oil." 

In  1852  Schlossberger  made  experiments  with  the 
pure  fatty  acids  and  demonstrated  their  freedom  from  poi- 
sonous properties.  These  experiments  have  been  verified 
repeatedly,  so  that  now  it  is  well  known  that  all  the  fatty 
acids  obtainable  from  cheese  are  devoid  of  poisonous 
properties. 

It  may  be  remarked  here,  that  there  is  every  probability 
that  the  poisonous  substance  was  present  in  the  extracts 
obtained  by  the  older  chemists.  Indeed,  we  may  say  that 
this  is  a  certainty,  since  the  administration  of  these  extracts 
to  cats  was,  in  some  instances  at  least,  followed  by  fatal 
result.  The  great  mass  of  these  extracts  consisted  of  fatty 
acids,  and  as  the  chemists  could  find  nothing  else  present, 
they  very  naturally  concluded  that  the  fatty  acids  them- 
selves constituted  the  poisonous  substance. 

Since  the  overthrow  of  the  fatty  acid  theory,  various 
conjectures  have  been  made,  but  none  worthy  of  considera- 
tion. 

We  make  the  following  quotations  from  some  of  the  best 
authorities  who  wrote  during  the  first  half  of  the  past 
decade  upon  this  subject : 

Hiller  says  :  "  Nothing  definite  is  known  of  the  nature 
of  cheese  poison.  Its  solubility  seems  established  from  an 
observation  by  Husemann,  a  case  in  which  the  poison  was 
transmitted  from  a  nursing  mother  to  her  child." 

Husemann  wrote  as  follows  :  "  The  older  investigations 
of  the  chemical  nature  of  cheese  poison,  which  led  to  the 
belief  of  putrefactive  cheese  acids  and  other  problematic 
substances,  are  void  of  all  trustworthiness,  and  the  dis- 
covery of  the  active  principle  of  poisonous  cheese  may  not 
be  looked  for  in  the  near  future,  on  account  of  the  proper 
animals  for  controlling  the  experiments  with  the  extracts, 


54  BACTERIAL    POISONS. 

as  dogs  can  eat  large  quantities  of  poisonous  cheese  without 
its  producing  any  effect." 

Brieger  stated  in  1885  :  "  All  kinds  of  conjectures  con- 
cerning the  nature  of  this  poison  have  been  formed,  but  all 
are  even  devoid  of  historical  interest;  because  they  are  not 
based  upon  experimental  investigations.  My  own  experi- 
ments toward  solving  this  question  have  not  progressed 
very  far." 

In  the  above  quotation  we  think  that  Brieger  has 
hardly  done  justice  to  the  work  of  Hunnefeld  and  Ser- 
turner.  Their  labors  can  hardly  be  said  to  be  wholly 
devoid  of  historical  interest,  and  they  certainly  did  employ 
the  experimental  method  of  inquiry. 

In  the  years  1883  and  1884  there  were  reported  to  the 
Michigan  State  Board  of  Health  about  three  hundred  cases 
of  cheese  poisoning.  As  a  rule,  the  first  symptoms  ap- 
peared within  from  two  to  four  hours  after  eating  the 
cheese.  In  a  few  the  symptoms  were  delayed  from  eight 
to  ten  hours  and  were  very  slight.  The  attending  physi- 
cians reported  that  the  gravity  of  the  symptoms  varied  with 
the  amount  of  cheese  eaten,  but  no  one  who  ate  of  the 
poisonous  cheese  wholly  escaped.  One  physician  reported 
the  following  symptoms :  "  Everyone  who  ate  of  the 
cheese  was  taken  with  vomiting,  at  first  of  a  thin,  watery, 
later  a  more  consistent  reddish-colored  substance.  At  the 
same  time  the  patient  suffered  from  diarrhoea  with  watery 
stools.  Some  complained  of  pain  in  the  region  of  the 
stomach.  At  first  the  tongue  was  white,  but  later  it  be- 
came red  and  dry,  the  pulse  was  feeble  and  irregular ; 
countenance  pale,  with  marked  cyanosis.  One  small  boy, 
whose  condition  seemed  very  critical,  was  covered  all  over 
the  body  with  bluish  spots." 

Dryness  and  constriction  of  the  throat  were  complained 
of  by  all.  In  a  few  cases  the  vomiting  and  diarrhoea  were 
followed  by  marked  nervous  prostration,  and  in  some  dila- 
tation of  the  pupils  was  observed. 

Notwithstanding  the  severity  of  the  symptoms  in  many, 
there  was  no  fatal  termination  among  these  cases,  though 
several  deaths  from  cheese  poisoning  in  other  outbreaks 


POISONOUS    CHEESE.  55 

have  occurred.  Many  of  the  physicians  at  first  diagnosed 
the  cases  from  the  symptoms  as  due  to  arsenical  poisoning, 
and  on  this  supposition  some  administered  ferric  hydrate. 
Others  gave  alcohol  and  other  stimulants  and  treated  upon 
the  expectant  plan. 

Vaughan,  to  whom  the  cheese  was  sent  for  aualysis, 
made  the  following  report:  "All  of  these  three  hundred 
cases  were  caused  by  eating  of  twelve  different  cheeses.  Of 
these,  nine  were  made  at  one  factory,  and  one  each  at  three 
other  factories.  Of  each  of  the  twelve  I  received  smaller 
or  larger  pieces.  Of  each  of  ten  I  received  only  small 
amounts.  Of  each  of  the  other  two  I  received  about 
eighteen  kilogrammes.  The  cheese  was  in  good  condition 
and  there  was  nothing  in  the  taste  or  odor  to  excite  sus- 
picion. However,  from  a  freshly  cut  surface  there  exuded 
numerous  drops  of  a  slightly  opalescent  fluid  which  red- 
dened litmus  paper  instantly  and  intensely.  Although,  as 
I  have  stated,  I  could  discern  nothing  peculiar  in  the  odor, 
if  two  samples,  one  of  good,  the  other  of  poisonous  cheese, 
were  placed  before  a  dog  or  cat,  the  animal  would  invari- 
ably select  the  good  cheese.  But  if  only  poisonous  cheese 
was  offered,  and  the  animal  was  hungry,  it  would  partake 
freely.  A  cat  was  kept  seven  days  and  furnished  only 
poisonous  cheese  and  water.  It  ate  freely  of  the  cheese 
and  manifested  no  untoward  symptoms.  After  the  seven 
days  the  animal  was  etherized  and  abdominal  section  was 
made.  Nothing  abnormal  could  be  found.  I  predicted, 
however,  in  one  of  my  first  articles  on  poisonous  cheese, 
that  the  isolated  poison  would  affect  the  lower  animals. 
As  to  the  truth  of  this  prediction  we  will  see  later. 

"  My  friend,  Dr.  Sternberg,  the  eminent  bacteriologist, 
found  in  the  opalescent  drops  above  referred  to  numerous 
micrococci.  But  inoculations  of  rabbits  with  these  failed 
to  produce  any  results. 

"At  first  I  made  an  alcoholic  extract  of  the  cheese.  After 
the  alcohol  was  evaporated  in  vacuo  at  a  low  temperature 
a  residue  consisting  mainly  of  fatty  acids  remained.  I  ate 
a  small  bit  of  this  residue,  and  found  that  it  produced  dry- 
ness of  the  throat,  nausea,  vomiting,  and  diarrhoea.     The 


56  BACTERIAL    POISONS. 

mass  of  this  extract  consisted  of  fats  and  fatty  acids,  and 
for  some  weeks  I  endeavored  to  extract  the  poison  from 
these  fats,  but  all  attempts  were  unsuccessful.  I  then  made 
an  aqueous  extract  of  the  cheese,  filtered  this,  and  drinking 
some  of  it,  found  that  it  also  was  poisonous.  But  after 
evaporating  the  aqueous  extract  to  dryness  on  the  water- 
bath  at  100°,  the  residue  thus  obtained  was  not  poisonous. 
From  this  I  ascertained  that  the  poison  was  decomposed  or 
volatilized  at  or  below  the  boiling-point  of  water.  I  then 
tried  distillation  at  a  low  temperature,  but  by  this  the 
poison  seemed  to  be  decomposed. 

"  Finally,  I  made  the  clear,  filtered  aqueous  extract, 
which  was  highly  acid,  alkaline  with  sodium  hydrate,  agi- 
tated this  with  ether,  removed  the  ether,  and  allowed  it  to 
evaporate  spontaneously.  The  residue  was  highly  poison- 
ous. By  re-solution  in  water  and  extraction  with  ether,  the 
poison  was  separated  from  foreign  substances.  As  the  ether 
took  up  some  water,  this  residue  consisted  of  an  aqueous 
solution  of  the  poison.  After  this  was  allowed  to  stand  for 
some  hours  in  vacuo  over  sulphuric  acid,  the  poison  sepa- 
rated in  needle-shaped  crystals.  From  some  samples  the 
poisoned  crystallized  from  the  first  evaporation  of  the  ether, 
and  without  standing  in  vacuo.  This  happened  only  when 
the  cheese  contained  a  comparatively  large  amount  of  the 
poison.  Ordinarily,  the  microscope  was  necessary  to  detect 
the  crystalline  shape.  From  sixteen  kilogrammes  of  one 
cheese,  I  obtained  about  0.5  gramme  of  the  poison,  and  in 
this  case  the  individual  crystals  were  plainly  visible  to  the 
unaided  eye.  From  the  same  amount  of  another  cheese  I 
obtained  only  about  0.1  gramme,  and  the  crystals  in  this 
case  were  not  so  large.  I  have  no  idea,  however,  that  by 
the  method  used  all  the  poison  was  separated  from  the 
cheese." 

To  this  ptomaine  Vauohan  has  given  the  name  tyro- 
toxicon  (rvpoQ,  cheese,  and  Tofjmov,  poison).  Its  chemistry 
will  be  discussed  in  a  subsequent  chapter. 

During  1887,  Wallace  found  tyrotoxicon  in  two 
samples  of  cheese  which  had  caused  serious  illness.  The 
first  of  these  came  from  Jeanesville,  Pa.,  and  the  symptoms 


POISONOUS    CHEESE.  57 

as  reported  to  Wallace  by  Doolittle,  who  had  charge 
of  the  cases,  were  as  follows:  "There  were  at  least  fifty 
persons  poisoned  by  this  cheese.  There  were  also  eight 
others  who  ate  of  the  cheese,  but  felt  no  unpleasant  effects ; 
whether  this  was  due  to  personal  idiosyncrasy,  or  to  an 
uneven  distribution  of  the  poison  throughout  the  cheese,  I 
am  unable  to  say. 

"  The  majority,  however,  comprising  fifty  or  sixty  per- 
sons, were  seized,  in  from  two  to  four  hours  after  eating  the 
cheese,  with  vertigo,  nausea,  vomiting,  and  severe  rigors, 
though  varying  in  their  order  of  appearance  and  in  severity 
in  different  cases.  The  vomiting  and  chills  were  the  most 
constant  and  severe  symptoms  in  all  the  cases,  and  were 
soon  followed  by  severe  pain  in  the  epigastric  region, 
cramps  in  the  feet  and  lower  limbs,  purging  and  griping 
pain  in  the  bowels,  a  sensation  of  numbness  or  pins  and 
needles,  especially  in  the  limbs,  and  lastly,  very  marked 
prostration,  amountiug  almost  to  collapse  in  a  few  cases. 

"The  vomit  at  first  consisted  of  the  contents  of  the 
stomach,  and  had  a  strong  odor  of  cheese ;  afterward  it 
consisted  of  mucus,  bile,  and  in  three  or  four  of  the  severer 
cases  blood  was  mixed  with  the  mucus  in  small  quantities. 
Microscopic  examination  of  the  same  was  not  made,  but  to 
the  eye  it  appeared  as  such.  The  vomiting  and  diarrhoea 
lasted  from  two  to  twelve  hours ;  the  rigors  and  muscular 
cramps,  one  to  two  hours.  The  diarrhceal  discharges,  at 
first  fecal,  became  later  watery  and  light  colored.  No 
deaths  occurred,  and  for  the  most  part  the  effects  were 
transient,  and  all  that  remained  on  the  following  day  were 
the  prostration  and  numbness ;  the  latter  occurred  in  about 
one-half  the  cases,  and  disappeared  in  from  one  to  three 
days. 

"Children,  as  a  rule,  seemed  to  suffer  less  than  adults, 
and,  of  course,  it  was  not  possible  to  elicit  as  definite  symp- 
toms from  them.  The  suddenness  of  the  attack  was 
remarked  by  all,  some  feeling  perfectly  well  until  the 
moment  of  attack.  Nor  did  the  symptoms  seem  to  be  in 
proportion  to  the  amount  of  cheese  taken  ;  some  of  the 
severest  cases  declared  they  had  not  eaten  more  than  a  cubic 


58  BACTERIAL    POISONS. 

inch  of  it.  One  of  the  severest  cases  was  about  six  and 
one-half  mouths  pregnant,  but  no  interference  with  preg- 
nancy occurred.  All  the  cheese  which  caused  the  sickness 
came  from  the  same  piece." 

The  second  sample  of  cheese  examined  by  Wallace 
came  from  Riverton,  N.  J.  This  outbreak  included  a 
smaller  number  of  persons,  all  of  whom  recovered. 

Wolff  has  detected  tyrotoxicon  in  cheese  which  poisoned 
several  persons  at  Shamokin,  Pa.  The  pores  of  this  cheese 
were  found  filled  with  a  grayish-green  fuugoid  growth, 
though  it  is  uot  supposed  that  this  fungus  was  connected  in 
any  way  with  the  poisonous  nature  of  the  cheese.  Tests 
were  made  for  mineral  poison  with  negative  results,  after 
which  tyrotoxicon  was  recognized  both  by  chemical  and 
physiological  tests.  "  A  few  drops  of  the  liquid  (extract), 
placed  on  the  tongue  of  a  young  kitten,  produced  prompt 
emesis  and  numerous  watery  dejections  with  evident  depres- 
sion and  malaise  of  the  animal.  A  larger  cat  was  similarly 
affected  by  it,  though  the  depression  and  malaise  were  not 
so  marked  nor  so  long  continued." 

Cheese  poisoning  caused  the  death  of  several  children  in 
the  neighborhood  of  Heiligenstadt,  in  1879,  and  there  were 
many  fatal  cases  from  the  same  cause  in  Pyrmont,  in  1878. 
Unfortunately  we  have  not  been  able  to  find  any  detailed 
account  of  either  the  symptoms  or  the  post-mortem  appear- 
ances in  these  cases. 

Ehrhart  has  published  the  history  of  some  cases  of 
poisoning  from  cheese,  of  which  the  following  is  an  abstract : 
The  family  of  a  workman,  consisting  of  eight  persons,  ate  for 
supper  600  grammes  (about  eighteen  ounces)  of  Limburger 
cheese.  The  rind  was  covered  with  a  heavy  mould,  while 
the  interior  had  become  fluid  from  putrefaction,  and  was  of 
bitter  taste.  Three  ate  only  of  the  mouldy  rind,  and  these 
remained  well.  The  next  morning,  the  five  who  had  eaten 
of  the  inner  portion  suffered  from  vertigo,  nausea,  vomiting, 
and  abdominal  pains ;  no  stool.  The  father  had  convulsive 
movements  of  all  the  extremities.  The  pupils  were  dilated, 
and  did  not  respond  to  light;  there  were  double  vision, 
cold  sweat,  skin  cyanotic,  abdomen  distended,  difficulty  in 


POISONOUS    CHEESE.  59 

swallowing,  delirium,  mild  trismus,  and  temperature  40°  C. 
(104°  F.).  The  temperature  of  the  mother,  on  account  of 
the  great  collapse,  was  subnormal.  She  had  no  convulsive 
movements,  but  there  was  prolonged  loss  of  consciousness. 
The  pulse  was  small  and  thready,  and  threatened  paralysis 
of  the  heart.  Recovery  was  very  slow.  The  others  suf- 
fered only  from  gastro-enteric  symptoms.  Ehrhardt 
discusses  the  question  as  to  whether  these  symptoms  were 
due  to  tyrotoxicon,  or  to  infection  with  microorganisms ; 
but  as  we  have  not  had  access  to  his  original  paper,  we  do 
not  know  what  his  conclusions  are.  However,  there  cannot 
be  much  doubt  that  in  those  cases  in  which  the  organism 
is  taken  into  the  alimentary  canal,  it  continues  the  elabora- 
tion of  its  poisonous  products. 

In  1890  Vaughan  made  the  following  additional  report 
on  poisonous  cheese  : 

"  During  the  past  two  or  three  years  we  have  received 
at  the  Hygienic  Laboratory  of  Michigan  University  a 
number  of  samples  of  cheese  which,  it  was  claimed,  had 
caused  nausea  and  vomiting  in  those  eating  of  them,  and 
in  which  we  were  unable  to  detect  tyrotoxicon.  Some  of 
these  samples  produced  vomiting  and  purging  in  cats  and 
dogs  to  which  the  cheese  was  fed  directly.  The  evidence 
that  these  samples  had  been  the  actual  cause  of  the  sickness 
among  the  people  who  had  eaten  of  them  was  thus  con- 
firmed by  the  experiments  upon  the  animals  ;  but  inasmuch 
as  we  were  unable  to  detect  the  poison,  we  were  compelled 
to  report  as  follows  : 

lt '  The  poisonous  character  of  the  cheese  has  been  proven 
by  experiments  upon  animals,  but  we  have  failed  to  demon- 
strate the  nature  of  the  poison.  Tyrotoxicon  could  not  be 
detected.' 

"  One  sample  of  this  class  was  found  by  Novy  to  be  very 
poisonous.  Some  of  this  cheese  was  covered  with  absolute 
alcohol,  and  after  standing  in  a  dish  for  some  weeks  the 
alcohol  was  allowed  to  evaporate,  then  100  grammes  of  the 
cheese  was  fed  to  a  young  dog  and  caused  its  death  within 
a  few  hours.  Sterilized  milk  to  which  a  small  bit  of  the 
cheese  was  added,  after  standing:  in  the  incubator  at  35°. 


60  BACTERIAL    POISONS. 

for  twenty-four  hours,  became  so  poisonous  that  100  c.  c.  of 
it  introduced  into  the  stomach  of  a  full-grown  cat  caused 
death.  JSTovy  made  plate  cultures  from  the  cheese  and 
from  the  spleen  and  liver  of  the  dead  animals,  and  suc- 
ceeded in  identifying  one  germ  as  common  to  both.  Ster- 
ilized milk  inoculated  with  a  pure  culture  of  this  germ,  and 
kept  in  the  incubator,  proved  fatal  to  cats.  But  with  the 
advent  of  cold  weather  the  germ  lost  its  toxicogenic  prop- 
erties, which  were  not  restored  by  subsequent  cultivation  in 
the  incubator. 

"  In  a  second  class  of  samples,  the  poisonous  character 
of  the  cheese  was  not  confirmed  by  direct  feeding.  Cats, 
rats,  and  dogs  were  fed  with  the  same  quantities  as  above, 
without  any  appreciable  effect.  The  report  made  upon  the 
samples  was  as  follows  : 

"  '  Animals  fed  upon  the  cheese  were  not  affected.  Tyro- 
toxicon  could  not  be  found.  The  sickness  in  the  people 
was  probably  due  to  some  other  cause/ 

"The  last  sentence  of  this  report  was  probably  wrong,  as 
will  be  shown  from  the  following  experiment.  Two  kilo- 
grammes of  a  cheese  of  this  class  was  extracted  repeatedly 
with  absolute  alcohol.  The  part  insoluble  in  alcohol  was 
then  extracted  with  water.  The  aqueous  extract,  after 
filtration,  was  allowed  to  fall  slowly  into  three  times  its 
volume  of  absolute  alcohol.  A  voluminous,  flocculent 
precipitate  resulted.  After  twenty-four  hours  the  super- 
natant fluid  was  decanted,  and  the  precipitate  was  dissolved 
in  water  and  re-precipitated  with  absolute  alcohol ;  then  it 
was  collected  and  speedily  dried  on  porous  plates.  A  small 
bit  of  this  precipitate  was  dissolved  in  water ;  and  forty 
drops  of  this  solution,  injected  under  the  skin  on  the  back 
of  cats,  produced  invariably  within  one  hour  vomiting  and 
purging.  After  the  partial  collapse  which  followed  the 
vomiting  and  purging,  and  which  was  evidenced  by  the 
animal  sitting  with  its  chin  resting  on  the  floor,  recovery 
gradually  followed.  The  same  amount  of  the  solution 
injected  into  the  abdominal  cavity  of  white  rats  rendered 
the  animals  within  ten  or  fifteen  minutes  perfectly  limp, 
and  the  only  evidence  of  life  observed  was  rapid  respiratory 


POISONOUS    CHEESE.  61 

movements.  The  rats  lay  upon  their  sides,  and  could  be 
handled  without  manifesting  any  attempt  at  movement. 
In  this  condition  some  died  after  three  or  four  hours,  while 
others,  after  lying  in  this  position  for  from  eighteen  to 
twenty-four  hours,  gradually  improved,  and  alter  some 
days  seemed  to  be  wholly  recovered. 

"  This  substance  belongs  to  the  so-called  poisonous  albu- 
mins. From  its  aqueous  solutions  it  is  not  precipitated  by 
heat  or  nitric  acid,  singly  or  combined.  Its  solutions 
respond  to  the  biuret  test.  It  is  not  precipitated  by  satura- 
tion with  sodium  sulphate,  nor  by  a  current  of  carbonic 
acid  gas  ;  therefore,  it  is  not  a  globulin.  It  is  precipitated 
by  saturation  with  ammonium  sulphate;  and  this  fact 
removes  it  from  the  peptones. 

"  That  animals  were  not  affected  when  fed  with  the 
whole  cheese  may  be  explained  by  the  supposition  that  they 
did  not  in  this  manner  get  enough  of  the  poison  to  affect 
them.  It  cannot  be  said  positively  that  the  samples  of 
cheese  of  the  first  class  mentioned  above  owe  their  poison- 
ous properties  to  this  substance.  We  have  not  had  the 
opportunity  of  testing  samples  of  this  class  since  the 
recognition  of  the  poisonous  proteid  in  those  of  the 
second  class.  Four  samples  of  the  latter  have  been  tested 
for  the  poisonous  albumin  with  positive  results. 

"  It  may  be  found  that  traces  of  this  poison  exist  in  all 
samples  of  green  cheese.     This  point  will  be  investigated. 

"  It  is  highly  probable  that  the  poisonous  effects  of  some 
samples  of  sausage  and  meat  are  due  to  similar  products  of 
bacterial  activity." 

In  reference  to  the  poisonous  proteids  in  cheese  and  other 
articles  of  food  the  following  interesting  questions  arise  : 
How  is  the  poisoning  explained  ?  Is  it  not  generally  sup- 
posed that  poisonous  proteids  are  not  absorbable  from 
mucous  membranes?  Mitchell  and  Reichert  showed 
that  the  venom  of  serpents  may  be  absorbed  from  mucous 
membranes;  especially  did  they  find  this  to  be  true  of  the 
poisonous  peptoue  of  the  cobra.  It  may  be,  however,  that 
the  bacteria,  which  are  in  the  cheese  and  to  which  the 
formation  of  the  poisonous  proteids  is  due,  find  their  way 

4 


62  BACTERIAL    POISONS. 

through  the  intestinal  walls  and  form  their  poisonous  pro- 
ducts within  the  spleen  and  other  organs.  The  fact  that 
Now  found  the  bacteria  in  the  spleen  and  liver  of  the 
animals  experimented  upon  confirms  this  view. 

Poisonous  Milk. — In  1885  Vaughan  found  tyrotoxi- 
con  in  milk  which  had  stood  in  a  well-stoppered  bottle  for 
about  six  months.  It  was  presumed  that  this  milk  was, 
when  first  obtained,  normal  in  composition,  but  since  this 
was  not  known  with  certainty,  the  following  experiments 
were  made  :  Several  gallon  bottles  were  filled  with  normal 
milk,  tightly  closed  with  glass  stoppers,  and  allowed  to 
stand  at  the  ordinary  temperature  of  the  room.  From  time 
to  time  a  bottle  was  opened  and  the  test  for  tyrotoxicon  was 
made.  These  tests  were  followed  by  negative  results  until 
about  three  months  after  the  experiment  was  begun.  Then 
the  poison  was  obtained  from  one  of  the  bottles.  The  coagu- 
lated milk  was  filtered  through  paper.  The  tiltrate,  which 
was  colorless  and  decidedly  acid  in  reaction,  was  rendered 
feebly  alkaline  by  the  addition  of  potassium  hydrate  and 
agitated  with  ether.  After  separation,  the  ethereal  layer 
was  removed  with  a  pipette,  passed  through  a  dry  filter- 
paper  in  order  to  remove  a  flocculent,  white  substance  which 
floated  in  it,  and  then  allowed  to  evaporate  spontaneously. 
If  necessary,  this  residue  was  dissolved  in  water  and  again 
extracted  with  ether.  As  the  ether  takes  up  some  water, 
there  is  usually  enough  of  the  latter  left  after  the  sponta- 
neous evaporation  of  the  ether  to  hold  the  poison  in  solu- 
tion, and  in  order  to  obtain  the  crystals  this  aqueous  solu- 
tion must  be  allowed  to  stand  for  some  hours  in  vacuo 
over  sulphuric  acid. 

From  one-half  gallon  of  the  milk  there  was  obtained 
quite  a  concentrated  aqueous  solution  of  the  poison  after 
the  spontaneous  evaporation  of  the  ether.  Ten  drops  of 
this  solution  placed  in  the  mouth  of  a  small  dog,  three 
weeks  old,  caused  within  a  few  minutes  frothing  at  the 
mouth,  retching,  the  vomiting  of  frothy  fluid,  muscular 
spasms  over  the  abdomen,  and  after  some  hours  watery 
stools.     The  next  day  the  dog  seemed  to  have  partially 


POISONOUS    MILE.  63 

recovered,  but  was  unable  to  retain  any  food.  This  condi- 
tion continuing  for  two  or  three  days  the  animal  was  killed 
with  chloroform.  JNTo  examination  of  the  stomach  was 
made. 

In  1886  Newton  and  Wallace  obtained  tyrotoxicon 
from  milk  and  studied  the  conditions  under  which  it  forms. 
Their  report  is  of  so  much  value  that  the  greater  part  of  it 
is  herewith  inserted. 

"  On  August  7th  twenty-four  persons,  at  one  of  the  hotels 
at  Long  Branch,  were  taken  ill  soon  after  supper.  At 
another  hotel,  on  the  same  evening,  nineteen  persons  were 
seized  with  the  same  form  of  sickness.  From  one  to  four 
hours  elapsed  between  the  meal  and  the  first  symptoms. 
The  symptoms  noticed  were  those  of  gastro-intestinal  irri- 
tation, similar  to  poisoning  by  any  irritating  material — 
that  is,  nausea,  vomiting,  cramps,  and  collapse  ;  a  few  had 
diarrhoea.  Dryness  of  the  throat  and  burning  sensation 
in  the  oesophagus  were  prominent  symptoms. 

"  While  the  cause  of  the  sickness  was  being  sought  for, 
and  one  week  after  the  first  series  of  cases,  thirty  persons 
at  another  hotel 'were  taken  ill  with  precisely  the  same 
symptoms  as  noticed  in  the  first  outbreak. 

"  When  the  news  of  the  outbreak  was  published  one  of 
us  immediately  set  to  work,  under  the  authority  of  the  State 
Board  of  Health,  to  ascertain  the  cause  of  the  illness.  The 
course  of  the  investigation  was  about  as  follows  : 

"  The  character  of  the  illness  indicated,  of  course,  that 
some  article  of  food  was  the  cause,  and  the  first  part  of  our 
task  was  to  single  out  the  one  substance  that  seemed  at 
fault.  The  cooking  utensils  were  also  suspected,  because 
unclean  copper  vessels  have  often  caused  irritant  poisoning. 
Articles  of  food,  such  as  lobsters,  crabs,  blue  fish,  and 
Spanish  mackerel,  all  of  which  at  times,  and  with  some 
persons  very  susceptible  to  gastric  irritation  have  produced 
toxic  symptoms,  were  looked  for,  but  it  was  found  that 
none  of  these  had  been  eaten  at  the  time  of  the  outbreak. 
The  cooking  vessels  were  examined,  and  all  were  found 
clean  and  bright,  and  no  evidence  of  corrosion  was  pre- 
sented. 


64  BACTERIAL    POISONS. 

"  Further  inquiry  revealed  the  fact  that  all  who  had 
been  taken  ill  had  used  rnilk  in  greater  or  less  quantities, 
aud  that  persons  who  had  not  partaken  of  milk  escaped 
entirely ;  corroborative  of  this,  it  was  ascertained  that 
those  who  had  used  milk  to  the  exclusion  of  all  other  food 
were  violently  ill.  This  was  prominently  noticed  in  the 
cases  of  infants  fed  from  the  bottle,  when  nothing  but  un- 
cooked milk  was  used.  In  one  case  an  adult  drank  about 
a  quart  of  the  milk,  and  was  almost  immediately  seized 
with  violent  vomiting  followed  by  diarrhoea,  and  this  by 
collapse.  Suffice  it  to  say,  that  we  were  able  to  eliminate 
all  other  articles  of  food  and  to  decide  that  the  milk  was  the 
sole  cause  of  the  outbreak. 

"  Having  been  able  to  determine  this,  the  next  step  was 
to  discover  why  that  article  should,  in  these  cases,  cause  so 
serious  a  form  of  sickness. 

"  The  probable  causes  which  we  were  to  investigate  were 
outlined  as  follows  :  (1)  Some  chemical  substance,  such  as 
borax,  boric  acid,  salicylic  acid,  sodium  bicarbonate,  sodium 
sulphate,  added  to  preserve  the  milk  or  to  correct  acidity. 
(2)  The  use  of  polluted  water  as  an  adulterant.  (3)  Some 
poisonous  material  accidentally  present  in  the  milk.  (4)  The 
use  of  milk  from  diseased  cattle.  (5)  Improper  feeding  of 
the  cattle.  (6)  The  improper  care  of  the  milk.  (7)  The 
development  in  the  milk  of  some  ferment  or  ptomaine, 
such  as  tyrotoxicon. 

"At  the  time  of  the  first  outbreak  we  were  unable,  un- 
fortunately, to  obtain  any  of  the  noxious  milk,  as  that  un- 
consumed  had  been  destroyed;  but  at  the  second  outbreak 
a  liberal  quautity  was  procured. 

"It  was  soon  ascertained  that  one  dealer  had  supplied 
all  the  milk  used  at  the  three  hotels  where  the  cases  of 
sickness  had  occurred.  His  name  and  address  having  been 
obtained,  the  next  step  in  the  investigation  was  to  inspect 
all  the  farms,  and  the  cattle  thereon,  from  which  the  milk 
was  taken.  We  also  learned  that  two  deliveries  at  the 
hotels  were  made  daily,  one  in  the  morning  and  one  in  the 
evening ;  that  the  milk  supplied  at  night  was  the  sole 
cause  of  the  sickness,  and  that  the  milk  from  but  one  of 


POISONOUS    MILK.  65 

the  farms  was  at  fault.  The  cows  on  this  farm  were  found 
to  be  in  good  health,  and,  besides  being  at  pasture,  were 
well  fed  with  bran,  middlings,  and  corn-meal. 

"  So  far  we  had  been  able  to  eliminate  as  causes  diseased 
cattle  and  improper  feeding,  and  we  were  then  compelled 
to  consider  the  other  possible  sources  of  the  toxic  material. 

"  While  the  inspection  of  the  farms  was  being  made,  the 
analysis  of  the  milk  was  in  progress.  The  results  of  this 
showed  that  no  chemical  substance  had  been  added  to  the 
milk,  that  it  was  of  average  composition,  that  no  polluted 
water  had  been  used  as  a  diluent,  and  that  no  poisonous 
metals  were  present.  This  result  left  us  nothing  to  con- 
sider but  two  probable  causes  :  improper  care  of  the  milk, 
and  the  presence  of  a  ferment. 

"As  to  the  former,  we  soon  learned  much.  The  cows 
were  milked  at  the  unusual  and  abnormal  hours  of  mid- 
night and  noon,  and  the  noon's  milking — that  which  alone 
was  followed  by  illness — was  placed,  while  hot,  in  the  cans, 
and  then,  without  any  attempt  at  cooling,  carted  eight  miles 
during  the  warmest  part  of  the  day  in  a  very  hot  mouth. 

"This  practice  seemed  to  us  sufficient  to  make  the  milk 
unpalatable,  if  not  injurious,  for  it  is  well  known  that  when 
fresh  milk  is  closed  up  in  a  tight  vessel  and  then  deposited 
in  a  warm  place,  a  very  disagreeable  odor  and  taste  are 
developed.  Old  dairymen  speak  of  the  animal  heat  as  an 
entity,  the  removal  of  which  is  necessary  in  order  that  the 
milk  shall  keep  well  and  have  a  pleasant  taste.  While  we 
do  not  give  this  thing  a  name,  we  are  fully  convinced  that 
milk  should  be  thoroughly  cured  by  proper  chilling  and 
aeration  before  it  is  transported  any  distance  or  sold  for 
consumption  in  towns  or  cities. 

"  This  opinion  is  based  on  a  study  of  the  methods  prev- 
alent among  experienced  dairymen,  who  ship  large  quanti- 
ties of  milk  to  our  great  cities.  The  usual  practice  is  to 
allow  the  milk  to  stand  in  open  vessels,  surrounded  by  ice 
or  cold  water,  for  from  eight  to  twelve  hours  before  trans- 
portation, and  when  placed  on  the  cars  it  has  a  temperature 
of  from  50°  to  60°  F.,  and  is  delivered  to  consumers  in  a 
perfectly  sweet  condition.     The  city  of  New  York  receives 


66  BACTEKIAL    POISONS. 

about  200,000  gallons  each  day  from  the  surrounding 
country,  and  much  of  it  brought  in  by  the  railroads  has 
been  on  the  cars  for  a  time  varying  from  six  to  twelve 
hours,  yet  we  seldom  hear  of  any  of  this  milk  undergoing 
the  peculiar  form  of  fermentation  set  up  in  the  Long 
Branch  milk.  We  may  account  for  this  by  assuming  that 
the  proper  care  of  the  milk  after  it  was  taken  from  the 
cow,  and  the  low  temperature  at  which  it  was  kept,  have 
prevented  the  formation  of  any  ferment ;  this  opinion 
seems  to  be  endorsed  by  all  dairymen  and  managers  of 
large  creameries  with  whom  we  have  consulted.  They  all 
agree  in  stating  that  milk  maintained  at  a  low  temperature 
can  be  kept  sweet  and  in  good  condition  for  many  days. 

"  We  have  dwelt  on  this  branch  of  our  topic  somewhat 
extensively,  because  we  are  fully  persuaded  that  the  im- 
proper care  of  the  milk  had  much  to  do  with  the  illness  it 
produced. 

"  The  results  of  our  inquiry  having  revealed  so  much, 
we  next  attempted  to  isolate  some  substance  from  the 
poisonous  milk,  in  order  that  the  proof  might  be  more 
evident.  A  quantity  of  the  milk  that  had  caused  sickness 
in  the  second  outbreak  was  allowed  to  coagulate,  was  then 
thrown  on  a  coarse  filter,  and  the  filtrate  collected.  This 
latter  was  highly  acid,  and  was  made  slightly  alkaline  by 
the  addition  of  potassium  hydrate.  This  alkaline  filtrate 
wras  now  agitated  with  an  equal  volume  of  pure,  dry  ether, 
and  allowed  to  stand  for  several  hours,  when  the  ethereal 
layer  was  drawn  oif  by  means  of  a  pipette.  Fresh  ether 
was  added  to  the  residuum,  then  agitated,  and,  when  sepa- 
rated, was  drawn  off  and  added  to  the  first  ethereal 
extract.  This  was  now  allowed  to  evaporate  spontane- 
ously, and  the  residue,  which  seemed  to  contain  a  small 
amount  of  fat,  was  treated  with  distilled  water  and  filtered, 
the  filtrate  treated  with  ether,  the  ethereal  solution  drawn 
oif  and  allowed  to  evaporate,  when  we  obtained  a  mass  of 
needle-shaped  crystals.  This  crystalline  substance  gave  a 
blue  color  with  potassium  ferricyanide  and  ferric  chloride, 
and  reduced  iodic  acid.  The  crystals,  when  placed  on  the 
tongue,  gave  a  burning  sensation.     A  portion  of  the  crys- 


POISONOUS    MILK.  67 

tals  was  mixed  with  milk  and  fed  to  a  cat,  when,  in  the 
course  of  half  an  hour,  the  animal  was  seized  with  retching 
and  vomiting,  and  was  soon  in  a  condition  of  collapse,  from 
which  it  recovered  in  a  few  hours. 

"  We  are  justified  in  assuming,  after  weighing  well  all 
the  facts  ascertained  in  the  investigation,  that  the  sickness 
at  Long  Branch  was  caused  by  poisonous  milk,  and  that 
the  toxic  material  was  tyrotoxicon. 

"  The  production  of  this  substance  was  no  doubt  due  to 
the  improper  management  of  the  milk — that  is,  too  long  a 
time  was  allowed  to  elapse  between  the  milking  and  the 
cooling  of  the  milk,  the  latter  not  being  attended  to  until, 
the  milk  was  delivered  to  the  hotel ;  whereas,  if  the  milk 
had  been  cooled  immediately  after  it  was  drawn  from  the 
cows,  fermentation  would  not  have  ensued,  and  the  result- 
ing material,  tyrotoxicon,  would  not  have  been  produced." 

In  the  same  year,  Schearer  found  the  same  poison  in 
the  milk  used  by,  and  the  vomited  matter  of,  persons  made 
sick  at  a  hotel  at  Corning,  Iowa. 

In  1887,  Firth,  an  English  army  surgeon  stationed  in 
India,  reported  an  outbreak  of  milk  poisoning  among  the 
soldiers  of  his  garrison.  From  the  milk  he  separated,  by 
Vaughan's  method,  tyrotoxicon.  He  also  obtained  tyro- 
toxicon from  milk  which  had  been  kept  for  some  months 
in  stoppered  bottles,  as  had  been  previously  done  by 
Vaughan.     (See  page  62.) 

In  1887,  Mesic  and  Vaughan  observed  four  cases  of 
milk  poisoning,  three  of  which  terminated  fatally,  and 
Novy  and  Vaughan  obtained  tyrotoxicon  from  the  milk, 
and  from  the  contents  of  the  intestine  in  one  of  the  fatal 
cases.     Vaughan  reports  these  cases  as  follows  : 

"  September  23, 1887, 1  was  visited  by  Dr.  A.  G.  Mesic, 
of  Milan,  Michigan,  who  informed  me  that  he  had  four 
members  of  a  family  under  his  charge,  all  of  whom  were 
seriously  ill  with  peculiar  symptoms  which  he  believed  to 
be  caused  by  tyrotoxicon.  Since  Dr.  Mesic  has  written 
out  for  me  the  history  of  these  cases,  I  will  insert  his  report 
in  full,  as  follows  : 

"  '  Saturday,  September  17,  while  passing  the  residence 


68  BACTERIAL    POISONS. 

of  S.  H.  Evans,  a  respectable  farmer,  I  was  called  in  to  see 
him.  I  found  him — a  man  of  about  fifty  years,  spare 
and  muscular — vomiting  severely,  with  flushed  face,  but 
with  a  temperature  of  96°  F.  There  was  marked  throb- 
bing of  the  abdominal  aorta ;  the  tongue  had  a  white, 
heavy  coating,  and  the  breathing  was  very  labored.  I  set 
to  work  with  "the  ordinary  remedies  to  allay  the  vomiting, 
which  had  already  continued  for  some  hours.  The  vomited 
matters  were  colored  with  bile.  Pupils  were  dilated,  and 
a  rash  resembling  that  of  scarlatina,  but  coarser,  covered 
the  chest,  forearms,  and  legs  below  the  knees,  while  the 
abdomen  and  thighs  remained  unaffected.  As  the  bowels 
had  not  been  moved  since  the  beginning  of  the  attack,  I 
administered  a  purgative  dose  of  calomel  with  a  little  podo- 
phyllin  and  rhubarb.  On  Sunday  a  small  stool  resulted. 
During  that  day  and  night,  and  the  following  day,  the 
retching  and  vomiting  continued.  Small  doses  of  carbolic 
acid  seemed  to  give  the  most  relief.  After  the  movement 
of  the  bowels  the  symptoms  were  somewhat  more  prom- 
ising ;  but  a  heavy  and  unfavorable  stupor  was  observable 
and  persistent. 

"  '  On  Sunday  the  coating  of  the  tongue  remained  very 
thick,  and  had  changed  to  a  dark  brown  color.  At  first  I 
thought  that  his  symptoms  indicated  a  depressed  condition, 
which  I  had  known  in  one  instauce  to  precede  typhoid 
fever.  However,  after  a  few  days,  I  concluded  that  I 
must  look  for  the  cause  of  the  condition  among  the  poi- 
sons ;  but  I  could  think  of  no  one  poison  which  would 
be  likely  to  produce  all  the  symptoms  observed.  During 
Monday,  Tuesday,  and  Wednesday,  there  was  but  little 
change,  and  the  treatment  was  continued. 

" '  On  Thursday  morning  I  found  the  son  Arthur,  a  lad 
of  eighteen  years,  strong  and  vigorous,  suffering  with  the 
same  symptoms,  only  in  a  more  violent  form.  After 
supper  on  Wednesday  evening  he  was  taken  with  nausea 
and  vomiting.  He  had  no  rash,  but  the  symptoms  were 
otherwise  identical  with  those  of  the  father,  except  in  being 
more  severe.     I  gave  a  cathartic,  which  acted  only  slightly. 

(< '  At]  my  evening  visit  I  found  Mrs,  Evans,  a  lady  of 


POISONOUS    MILK.  09 

about  forty-five,  previously  in  good  health,  with  the  same 
symptoms.  Iu  this  case  the  stupor  was  more  marked  from 
the  first.  I  was  unable  at  any  time  to  obtain  any  cathartic 
action  in  this  case.  Copious  enemata  of  warm  water  were 
used,  but  succeeded  only  in  washing  some  hardened  lumps 
from  the  rectum.  By  this  time  I  had  concluded  that  the 
poison  was  most  likely  tyrotoxicon. 

" '  On  Friday  morning  the  only  remaining  member  of 
the  family  at  home,  Miss  Alma,  sixteen  years  of  age,  was 
affected  in  the  same  way  as  the  others.  On  that  day  I 
went  to  Ann  Arbor,  and  gave  a  history  of  the  cases  so  far 
to  Dr.  Vaughan,  who,  from  the  symptoms,  thought  that 
my  diagnosis  was  most  probably  correct,  and  he  advised 
with  me  as  to  treatment,  which  I  carried  out.  I  gave  two 
grains  of  sodium  salicylate  every  four  hours,  and  used  small 
doses  of  the  tonics  and  stimulants,  quinine,  mix  vomica, 
digitalis,  whiskey,  and  the  aromatic  spirits  of  ammonia. 
On  Saturday  the  symptoms  in  all  remained  unimproved, 
and  in  the  mother  and  son  the  stupor  and  labored  breath- 
ing grew  more  marked. 

"  '  On  Sunday,  I  again  went  to  Ann  Arbor,  and  brought 
Dr.  Vaughan  with  me  to  see  the  patients.  The  tempera- 
ture of  the  mother  on  Sunday  was  as  low  as  94°  F.,  and 
that  of  the  son  95°  F.  Dr.  "Vaughan  agreed  with  me  as  to 
diagnosis  and  treatment.  Sunday  evening  the  patients 
were  all  removed  to  the  house  of  a  neighbor,  about  forty 
rods  distant  (the  reasons  for  this  will  be  given  later).  Dr. 
Vaughan  and  I  both  expressed  the  fear  that  the  mother, 
and  possibly  the  son,  would  not  live  through  the  night. 
Both  of  these  rapidly  grew  worse,  and  the  son  died  at  7.45 
a.m.  and  the  mother  at  4  p.m.,  Monday. 

"  '  During  Monday  the  daughter  rapidly  grew  worse,  and 
at  the  time  of  her  mother's  death  could  not  be  aroused,  and 
practically  she  remained  unconscious  from  that  time  on. 
The  father  was  very  w^eak,  but  retained  his  consciousness 
all  the  time.  Convulsive  movements  of  the  limbs  had 
been  noticed  in  the  son,  but  not  in  the  mother.  These 
now  became  more  marked  in  the  daughter,  who  remained 

4* 


70  BACTERIAL    POISONS. 

in  the  heavy  stupor,  with  labored  breathing,  until  5  p.m. 
Thursday,  when  she  died. 

" '  Mr.  Evans  has  slowly  improved,  and  now,  October 
18th,  is  able  to  walk  about  the  room.  The  sodium  sali- 
cylate, even  in  the  small  doses  used,  seemed  to  cause  severe 
headache ;  so  apparent  was  this  that  the  drug  was  discon- 
tinued, and  drop  doses  of  amyl  nitrite,  given  every  hour, 
seemed  to  relieve  the  pain  in  the  head.  His  temperature 
remained  below  the  normal  until  Thursday,  October  14th, 
when  it  reached  the  normal.  After  this  it  was  found  once 
as  high  as  99.5°  F.,then  99°  F.,theu  again  normal,  where 
it  remains. 

"  'AH  complained  of  a  burning  constriction  in  the  throat, 
and  difficulty  in  swallowing,  and  all,  as  long  as  they  were 
conscious,  frequently  called  for  ice.  Iu  all  the  pulse  was 
rapid  and  feeble,  and  death  seemed  to  result  from  failure 
of  the  heart.  Those  who  died  voided  urine  involuntarily, 
while  Mr.  Evans  passed  small  quantities  frequently,  and 
for  this  buchu  and  uva  ursa  were  given.  During  his  con- 
valescence small  doses  of  morphine  were  given,  as  he  was 
unable  to  sleep,  and  became  very  restless.  He  is  now 
taking  teaspoonful  doses  of  the  elixir  of  calisaya  and  iron 
every  four  hours.' 

"  As  stated  above  by  Dr.  Mesic,  I  first  saw  these  patients 
Sunday,  September  25th.  On  a  sofa  in  the  room  we  found 
the  daughter,  Alma.  She  had  been  vomiting  during  the 
day,  and  seemed  much  exhausted.  She  was  not  inclined  to 
talk,  and  seemed  to  be  in  a  stupor,  though  when  spoken  to 
she  responded  rationally.  Her  pupils  were  slightly  dilated, 
her  tongue  coated,  her  pulse  120  and  weak,  her  face  flushed, 
and  a  violent  throbbing  could  be  felt  over  the  abdomen, 
which  was  retracted.     Her  temperature  was  96°  F. 

"  In  another  room  were  the  father,  mother,  and  son,  two 
of  them  dying.  The  father  was  rational,  and  talked  with 
some  freedom  when  I  asked  as  to  the  kind  of  food  they 
had  been  eating,  etc.  His  pupils  were  normal.  His  face 
could  not  be  said  to  present  any  peculiar  feature.  His 
pulse  was  rapid,  breathing  somewhat  labored,  and  the 
throbbing  of  the  abdominal  aorta  was  plainly  felt.     The 


POISONOUS    MILK.  71 

abdomen  was  retracted,  and  there  was  no  pain  on  pressure. 
He  complained  of  a  burning  constriction  of  the  throat, 
swallowed  with  difficulty,  and  said  that  his  throat  and 
stomach  felt  as  though  they  were  on  fire. 

"  The  mother  lay  perfectly  still  with  eyelids  closed,  as  if 
in  a  deep  sleep.  Her  pulse  was  rapid,  her  face  had  a  livid 
flush,  her  breathing  was  about  35  per  minute,  and  labored. 
The  skin  was  cool,  but  neither  abnormally  moist  nor 
specially  dry  and  harsh.  She  could  not  be  aroused.  In 
fact,  she  was  comatose. 

"  The  son  rolled  uneasily  from  one  side  of  the  bed  to  the 
other.  His  breathing,  also,  was  very  labored.  His  eyelids 
were  closed,  and  the  pupils  were  markedly  dilated — did  not 
respond  to  light.  He  could  not  be  aroused.  In  mother 
and  son,  as  well  as  in  father  and  daughter,  the  abdomen 
was  retracted,  and  the  throbbing  of  the  abdominal  aorta 
was  easily  felt. 

"  Now,  to  what  were  these  symptoms  due  ?  They  were 
certainly  those  of  some  poison.  Dr.  Mesic  had  brought 
me  some  of  the  vomited  matter,  which  I  tested  thoroughly 
for  mineral  poisons,  with  negative  results.  The  symptoms 
certainly  were  not  those  of  morphine,  strychnine,  digitalis, 
or  aconite.  They  did  have  some  resemblance  to  those  of 
belladonna,  but  yet  they  were  not  the  symptoms  of  bella- 
donna. The  pupils  were  not  as  widely  dilated  as  they 
would  be  in  belladonna  poisoning.  There  was  in  none  of 
these  persons  the  active  delirium  of  belladonna  poisoning. 
There  was  no  picking  at  the  clothing,  no  grasping  of  imag- 
inary objects  in  the  air,  no  hallucinations  of  vision.  Surely 
it  could  not  be  any  vegetable  alkaloid  with  which  I  was 
familiar. 

"  On  the  other  hand,  we  know  that  nausea,  vomiting, 
headache,  dilatation  of  the  pupil,  rapid  pulse,  heavy  breath- 
ing, constipation,  and  great  prostration,  with  stupor,  do 
occur  in  cases  of  poisoning  with  certain  ptomaines.  There- 
fore we  began  to  look  for  conditions  which  would  be  favor- 
able for  the  production  of  putrefactive  alkaloids.  These 
conditions  we  were  not  long  in  finding. 

"  The  family,  which  consisted  of  the  four  persons  sick,  and 


72  BACTERIAL    POISONS. 

of  a  daughter  about  twenty  years  of  age,  who  was  away 
from  home  at  the  time  when  the  others  were  taken  ill, 
and  for  some  months  before  that  time,  was  evidently  a 
tidy  one.  This  was  shown  by  their  personal  appear- 
ance, and  by  the  clothing  and  bedding.  But  the  house 
in  which  they  lived  was  very  old,  and  very  much 
decayed.  Mr.  Evans  had  purchased  the  farm  six  years 
ago ;  and  for  some  three  years  past,  at  least,  they  had  been 
troubled  every  now  and  then,  one  or  more  of  the  family, 
with  nausea  and  vomiting,  followed  by  more  or  less  prostra- 
tion. But  in  no  instance,  up  to  the  present  illness,  had  the 
symptoms  been  sufficient  to  cause  them  to  summon  a  physi- 
cian. The  family  had  worked  hard  in  order  to  pay  for  the 
farm,  and  had  determined  to  make  the  old  house  do  until 
they  were  out  of  debt.  Even  before  this  family  had  moved 
to  the  farm,  the  house  had  been  known  among  the  neigh- 
bors as  an  unhealthy  one,  and  there  had  been  much  sick- 
ness and  a  number  of  deaths  among  its  former  tenants. 

"  The  house  is  a  frame  one,  and  one  of  the  neighbors  said 
to  me  that  it  was  an  old  house  when  he  came  to  the  neigh- 
borhood thirty-seven  years  ago.  It  consists  of  two  rooms 
on  the  ground-floor,  with  attic  rooms  above.  The  frame 
rests  upon  four  large  logs  or  sills,  which  lie  directly  upon 
the  ground,  and  are  thoroughly  rotten.  There  is  no  cellar 
under  any  part  of  the  house.  From  the  front,  at  least,  the 
surface  slopes  toward  the  house,  and  the  rain-water  runs 
under  it.  In  the  floor  of  one  room  a  trap-door  had  been 
placed,  and  directly  under  this  a  small  excavation  had  been 
made  for  the  purpose  of  collecting  the  rain-water  when  it 
accumulated  under  the  house.  Although  this  pit  was  dry 
at  the  time  of  our  examination,  its  sides  and  bottom  were 
marked  with  cray-fish  holes,  showing  that  water  had  stood 
in  it.  The  floor  was  laid  of  unjointed  boards,  and  every 
time  that  it  was  swept  much  of  the  filth  fell  through  the 
cracks,  and  every  time  that  the  tidy  housewife  scoured  and 
mopped  the  floor,  the  water,  carrying  with  it  the  filth,  ran 
through  the  crevices,  and  thus  the  conditions  most  favorable 
for  putrefactive  changes  were  brought  into  existence  and 
maintained. 


POISONOUS    MILK.  73 

"  One  corner  of  one  of  the  rooms  had  been  transformed 
into  a  small  room,  or  buttery,  as  it  was  called,  and  in  this, 
on  shelves,  the  food  was  kept.  On  account  of  the  more 
frequent  scouring  demanded  by  that  part  of  the  floor 
enclosed  in  this  buttery,  the  boards  had  rotted  away,  and  a 
second  layer  of  boards  had  been  placed  over  the  original 
floor.  Between  these  two  floors  we  found  a  great  mass  of 
moist,  decomposing  -matter,  the  accumulations  of  years, 
which  the  broom  could  not  reach.  When  this  floor  was 
taken  up,  a  peculiar,  nauseating  odor  was  observable,  and 
was  sufficient  to  produce  nausea  and  vomiting  in  one  of  the 
persons  engaged  in  the  examination.  Some  of  the  dirt 
from  beneath  the  floor,  and  some  of  that  which  had  accumu- 
lated beneath  the  boards  in  the  buttery,  were  taken  for 
further  study. 

"  The  condition  of  the  house  was  supposed  to  be  unfavor- 
able to  the  patients,  and  for  this  reason  they  were  moved, 
as  Dr.  Mesic  has  stated,  to  the  house  of  a  neighbor.  Of 
course,  thorough  examination  of  the  house  was  not  made 
until  the  patients  had  been  removed. 

"  Special  inquiry  was  now  made  concerning  the  food  used 
by  this  family.  They  had  been  living  very  simply.  They 
lived  upon  bread,  butter,  milk,  and  potatoes,  with  coffee 
and  ripe  fruit.  They  had  eaten  no  canned  foods  for  months. 
They  ate  but  little  meat.  Occasionally  a  chicken  was  killed 
and  served,  aud  rarely,  some  fresh  meat  was  obtained  from 
the  village.  During  the  week  in  which  they  were  taken  ill, 
all  the  meat  used  consisted  of  slices  from  a  piece  of  bacon, 
the  only  meat  which  was  kept  in  the  house,  and  a  chicken. 
None  of  the  latter  remained,  but  the  bacon  was  examined. 
It  seemed  in  perfect  condition,  and  contained  no  trichinse. 
Moreover,  as  has  been  seen  from  the  history  of  the  cases, 
all  the  members  of  the  family  were  not  made  sick  by  any 
one  meal,  but  the  opportunity  of  obtaining  the  poison  must 
have  been  present  for  some  time.  Moreover,  the  fact  that 
previous  similar,  but  less  severe,  attacks  had  occurred  at 
intervals  for  the  past  three  years,  convinced  us  that  the 
poison  must  owe  its  origin  to  some  long-existing  condition. 

"  The  drinking-water  supply  was  also  investigated.    The 


74  BACTERIAL    POISONS. 

water  was  obtained  from  a  shallow  well,  and  some  of  it  was 
taken  for  analysis.  But  several  families  had  for  years  used 
water  from  this  well,  and  had  remained  healthy. 

"  The  milk  used  by  the  family  was  studied.  Of  course, 
we  could  get  none  of  that  which  had  been  used  before  the 
members  of  the  family  were  stricken  down.  As  soon  as  he 
made  the  diagnosis  of  tyrotoxicon  poisoning,  Dr.  Mesic 
ordered  the  discontinuance  of  the  use  of  milk,  not  only  with 
the  sick,  but  he  forbade  the  daughter,  who  had  returned, 
and  any  of  the  visitors  using  it.  Mr.  Evans  owned  four 
milch  cows,  and  they  were  supplied  with  fair  pasturage  and 
abundant  water.  The  greater  part  of  the  milk  was  placed 
in  tin  cans  which  were  set  in  a  wooden  trough  in  the  yard, 
and  surrounded  by  cold  water.  The  covers  to  the  cans 
were  arranged  so  that  the  air  could  have  free  access  to  the 
milk,  and  were  left  in  this  position  until  the  milk  was 
thoroughly  cooled.  Indeed,  the  cans  were  furnished  by  a 
creamery  company,  which  followed  the  directions  which  I 
have  previously  given  for  the  care  of  milk.  On  his  first 
visit  to  me,  Dr.  Mesic  brought  some  of  the  milk  from  one 
of  these  cans.  This  I  examined,  but  failed  to  find  tyro- 
toxicon in  it, 

"  However,  the  family  did  not  drink  any  of  the  milk  from 
the  cans.  That  which  they  did  use  was  kept  in  the  buttery 
which  I  have  described.  Here  it  stood  upon  a  shelf,  and 
some  members  of  the  family,  at  least,  were  in  the  habit  of 
drinking  from  it  between  meals.  This  was  especially  true, 
it  is  said,  of  the  son.  He  would  frequently  come  from  his 
work  in  the  fields,  go  into  the  buttery  and  drink  a  glass  or 
more  of  the  milk.  Mr.  Evans  states  that  he  frequently 
observed  that  the  taste  of  the  milk  was  not  pleasant.  On 
my  first  visit  to  the  premises  I  advised  that  some  of  the 
milk  should  be  taken  from  the  cans,  allowed  to  stand  in  the 
buttery  over  night,  and  be  sent  to  me  the  next  day.  This 
was  done,  and  in  this  milk  we  found  tyrotoxicon,  not  only 
by  the  employment  of  chemical  tests,  but  by  poisoning  a 
kitten  with  it. 

"On  the  death  of  the  mother  and  son,  Dr.  Mesic  asked  for 
a  post-mortem,  but  the  friends  objected,  and  the  undertaker 


POISONOUS    MILK.  75 

used  an  arsenical  embalming  fluid,  so  that,  although  consent 
was  subsequently  obtained,  it  was  decided  that  the  exami- 
nation would  be  so  vitiated  as  to  be  worthless.  On  the 
death  of  the  daughter,  the  coroner  summoned  a  jury  and 
held  an  inquest.  The  post-mortem  was  conducted  by  Dr. 
George  A.  Hendricks,  in  the  presence  of  the  jury  and 
several  physicians  who  had  been  invited.  Dr.  Hendricks 
has  kindly  furnished  me  with  his  report,  which  I  present 
here  iu  full : 

"  The  autopsy  was  held  fifteen  hours  after  death.  The 
abdominal  viscera  were  first  examined.  The  great  omen- 
tum was  small,  in  normal  position,  covering  the  small 
intestine.  The  small  intestine  was  moderately  distended 
with  flatus.  The  jejunum  was  ashy-green  in  color ;  the 
ileum  purplish -green.  About  eighteen  inches  from  the  ter- 
mination of  the  ileum  was  found  a  diverticulum  two  inches 
in  length.  The  small  intestine  contained  very  little  ali- 
mentary matter.  The  vermiform  appendix  was  free,  con- 
tained some  small  fecal  lumps,  and  showed  no  evidence  of 
inflammation.  The  csecum,  ascending,  transverse,  and 
descending  colon  were  empty  and  their  circular  fibres  were 
tightly  constricted,  except  at  intervals  where  the  intestine 
was  distended  with  gas.  The  sigmoid  flexure  was  moder- 
ately distended  with  gas,  and  the  rectum  contained  small 
bits  of  fecal  matter.  The  stomach  was  somewhat  contracted 
and  lay  wholly  upon  the  left  side  of  the  median  line.  It 
coutained  a  few  ounces  of  fluid.  Its  extremities  were  ligated 
and  the  organ  removed.  The  mucous  membrane  of  the 
stomach  and  intestine  were  not  examined  until  they  reached 
the  chemist.  The  duodenum  was  distended  with  flatus. 
The  liver  was  normal  in  size  and  appearance.  The  gall- 
bladder contained  about  one  ounce  of  bile.  The  spleen  was 
normal.  One-half  ounce  of  fluid  deeply  stained  with  blood 
was  found  in  Douglas's  cul-de-sac.  The  uterus,  Fallopian 
tubes,  and  ovaries  were  deeply  congested.  The  left  ovary 
was  enlarged  and  presented  on  its  posterior  surface  a  hemor- 
rhagic spot,  oval,  about  one-half  line  in  length,  and  several 
other  less  distinct  ones.  The  right  ovary  was  normal  in 
size  and  showed  numerous  Graafian  scars.     The    ureters 


76  BACTERIAL    POISONS. 

and  bladder  were  normal ;  the  latter  contained  a  small 
amount  of  urine.  The  peritoneum,  pancreas,  and  kidneys 
were  perfectly  normal. 

"  The  thoracic  cavity  was  next  opened.  The  lungs  were 
normal ;  there  was  about  one-half  ounce  of  free  serum  in 
the  left  pleural  cavity ;  none  in  the  right.  Pericardium 
normal ;  right  auricle  in  diastole ;  left  auricle  and  both 
ventricles  in  systole. 

"  The  dura  mater  showed  venous  congestion ;  the  arach- 
noid, normal ;  the  pia  mater,  congested.  On  the  surface 
of  the  centrum  ovale,  small  drops  of  blood  oozed  from  the 
divided  vessels.  The  large  veins  of  the  velum  interposi- 
tum  were  distended.  Third  and  fourth  ventricles  were 
slightly  distended  with  serous  fluid,  but  the  walls  were 
normal.  There  seemed  to  be  slight  softening  of  the  optic 
thalami.  The  sub-arachnoid  fluid  was  about  twice  the  nor- 
mal quantity. 

"  On  examination  of  the  mucous  membrane  of  the 
stomach  and  intestine  in  the  presence  of  the  chemist,  Prof. 
A.  B.  Prescott,* nothing  abnormal  could  be  found.  The 
membrane  was  stained  with  bile,  but  there  was  not  the 
slightest  redness.  The  solitary  glands  were  distinct,  but 
not  at  all  inflamed.     Peyer's  patches  were  normal. 

"It  will  be  seen  that  there  existed  no  lesion  which  would 
account  for  the  death.  The  venous  congestion  observed  in 
the  brain  would  follow  from  failure  of  the  heart. 

"  Some  of  the  post-mortem  appearances  bore  a  striking 
resemblance  to  those  which  I  had  observed  in  cats  poi- 
soned with  tyrotoxicon.  This  was  especially  noticeable  in 
the  condition  of  the  mucous  membrane  of  the  stomach  and 
intestine.  Tyrotoxicon  produces  the  symptoms  of  a  gas- 
trointestinal irritant,  but  not  the  lesions.  The  contraction 
of  the  circular  fibres  of  the  intestine,  which  undoubtedly 
caused  the  constipation,  I  had  also  observed  in  cats  that 
died  from  tyrotoxicon  poisoning  without  either  vomiting 
or  stool.1     The  action  of  this  poison  upon  the  stomach  and 

1  Marsh  reports  a  case  in  which  the  symptoms  resembled  very  closely 
those  of  rapidly  perforating  typhlitis,  but  the  post-mortem  examination 
showed  absolutely  no  evidence  of  this  disease  or  of  peritonitis.     In  fact  the 


POISONOUS    MILK.  77 

intestine  must  be  through  the  nervous  system.  Small 
doses  cause  both  vomiting  and  purging,  while  after  large 
doses  vomiting  may  be  impossible,  and  obstinate  constipa- 
tion may  exist.  Both  the  vomiting  and  purging  after 
small  doses  are  undoubtedly  due  in  part  to  increased 
activity  of  the  circular  fibres  of  the  muscular  coats,  induced 
through  the  nerves  ;  and  the  inability  to  vomit,  and  the 
constipation,  one  or  both  of  which  may  be  observed  after 
large  doses  of  the  poison,  are  due  to  spasm  of  the  same 
muscles,  induced  in  the  same  manner. 

"  Prof.  A.  B.  Prescott  was  requested  by  the  coroner  to 
analyze  the  material  for  mineral  and  vegetable  poisons. 
He  made  analyses  of  the  stomach  and  part  of  its  contents, 
and  a  portion  of  the  liver.  His  results  were  wholly  nega- 
tive. 

"  Novy  tested  a  cold-water  extract  of  the  finely  divided 
intestine  for  ptomaines.  The  fluid,  which  was  acid  in 
reaction,  was  filtered,  then  neutralized  with  sodium  bi- 
carbonate, and  shaken  with  ether.  The  ether,  after 
separation,  was  removed,  and  allowed  to  evaporate  spon- 
taneously. The  residue  was  dissolved  in  water,  and 
extracted  again  with  ether.  This  ether  residue  gave  the 
chemical  reactions  for  tyrotoxicon,  and  a  portion  of  it  was 
administered  to  a  kitten  about  two  months  old.  Within 
half  an  hour  after  the  administration  the  kitten  began  to 
retch,  and  soon  it  vomited.  Within  the  next  three  hours 
it  was  noticed  to  vomit  as  many  as  five  times.  The  breath- 
ing became  rapid  and  labored.  The  animal  sat  with  its 
head  down,  and  seemed  greatly  prostrated.  The  pupils 
were  examined,  but  could  not  be  said  to  be  dilated.  There 
was  no  purging.  The  retching  and  heavy  breathing,  with 
evidences  of  prostration,  continued  more  or  less  marked 
for  two  days,  after  which  the  animal  slowly  improved. 

"  A  quantity  of  fresh  milk  was  divided  into  five  por- 
tions of  one  quart  each,  placed  in  quart  bottles  which  had 

only  abnormality  found  in  the  intestines  consisted  of  the  contraction  ot  the 
circular  fibres  of  the  transverse  and  descending  colon.  Marsh  believes 
that  this  was  a  case  of  ptomaine  poisoning. 


78  BACTERIAL    POISONS. 

been  thoroughly  cleansed,  and  treated  in  the  following 
manner : 

"  No.  1  consisted  of  the  milk  only,  and  was  employed 
as  a  control  test. 

"No.  2  was  mixed  with  a  drachm  of  vomited  matter. 

''No.  3  was  treated  with  a  portion  of  the  contents  of 
the  stomach. 

"  No.  4  was  treated  with  an  aqueous  extract  of  the  in- 
testine. 

"  No.  5  was  treated  with  a  small  portion  of  the  soil 
which  had  been  taken  from  the  floor  of  the  buttery,  stirred 
up  with  water. 

"  These  bottles  were  placed  in  an  air-bath,  and  kept  at  a 
temperature  of  from  25°  to  30°  C.  for  twenty-four  hours. 
Then  each  was  tested  for  ptomaines.  No.  1  yielded  no 
tyrotoxicon,  while  all  of  the  others  contained  this  poison. 
The  tests  were  both  chemical  and  physiological.  All  of 
the  samples  yielded  a  non-poisonous  base  when  treated 
according  to  Brieger's  method,  and  the  same  substance  was 
obtained  from  perfectly  fresh  milk.  It  is  most  probably 
formed  by  the  action  of  the  heat  and  reagents  employed  in 
this  method.  This  base  was  obtained  in  crystalline  form, 
and  several  portions  of  it  were  administered  to  kittens 
without  any  effect.  The  further  study  of  this  body  will 
be  of  interest  to  toxicologists,  because  it  gives  many  of  the 
general  alkaloidal  reactions.  At  first  w7e  supposed  it  to  be 
Brieger's  neuridine,  and  this  supposition  may  still  be  cor- 
rect, but,  as  we  obtained  it,  it  gave  some  reactions  which 
are  not  given  by  neuridine.  Further  investigations  will 
be  made  on  this  point. 

"  Tyrotoxicon  was  obtained  from  the  filtered  milk  by 
two  methods:  (1)  The  one  which  we  have  previously  used, 
and  which  consists  in  neutralizing  the  filtered  milk  with 
sodium  bicarbonate,  and  extracting  with  ether.  That  por- 
tion of  the  poison  employed  in  the  physiological  tests  was 
obtained  in  this  way,  and  in  order  to  be  sure  that  no  poison 
came  from  the  ether,  the  extract  from  the  milk  to  which 
nothing  had  been  added  was  given  to  a  kitten,  and  was 
found  to  produce  no  effect.     (2)  The  filtrate  from  the  milk 


POISONOUS    ICE-CEEAM.  79 

was  heated  to  70°  C.  (158°  F.)  (tyrotoxicou  decomposes  at 
91°  C.  (195.8°  F.))  for  some  minutes,  and  filtered.  This 
filtrate,  which  Avas  perfectly  clear,  was  treated  with  a  small 
quantity  of  nitric  acid  in  order  to  convert  the  tyrotoxicon 
into  a  nitrate,  then  pure  potassium  hydrate  in  the  solid 
form  was  added  until  the  solution  was  strongly  alkaline. 
This  solution  was  concentrated  so  far  as  it  could  be  on  the 
water-bath.  (The  potassium  compound  of  tyrotoxicon  is 
not  decomposed  below  130°  C.  (234°  F.).)  The  dark 
brown  residue,  after  cooling,  was  examined  with  the  micro- 
scope and  found  to  contain  the  crystalline  plates  of  tyro- 
toxicon-potassinm  hydrate,  along  with  the  prisms  of  potas- 
sium nitrate.  The  former  was  separated  from  the  latter 
by  extraction  with  absolute  alcohol  and  filtration.  The 
alcohol  was  evaporated  to  dryness  on  the  water-bath,  and 
the  residue  again  extracted  with  absolute  alcohol.  From 
this  alcoholic  solution  tyrotoxicon  was  precipitated  with 
ether.  The  precipitate  was  decomposed  by  adding  acetic 
acid  and  heating,  the  tyrotoxicon  being  broken  up  into 
nitrogen  and  phenol.  The  phenol  was  recognized  by  pre- 
cipitation with  bromine  water,  and  by  other  well-known 
tests. 

"  On  October  8th,  the  coroner's  inquest,  which  had  been 
adjourned  after  the  post-mortem  in  order  to  await  the  re- 
sults of  the  analysis,  was  resumed,  and  after  hearing  the 
testimony  in  accordance  with  the  above  stated  facts,  the 
jury  returned  a  verdict  of  death  from  poisoning  with  tyro- 
toxicon." 

Cammajst  reports  twenty-three  cases  of  milk  poisoning 
which  he  attributes  to  tyrotoxicon,  although  this  poison 
could  not  be  found  in  the  milk.  It  may  be  that  the  active 
agent  present  belongs  to  the  bacterial  proteids. 

Kinnictjtt  has  isolated  tyrotoxicon  from  milk  which 
had  been  kept  for  some  hours  in  an  unclean  vessel. 

Poisonous  Ice-cream. — In  1886,  Yaughan  and 
Novy  obtained  tyrotoxicon  from  a  cream  which  had 
seriously  affected  many  person  at  Lawton,  Michigan. 
Vanilla  had  been  used  for  flavoring,  and  it  was  supposed 


80  BACTEEIAL    POISONS. 

that  the  ill-effects  were  due  to  the  flavoring.  This  belief 
was  strengthened  by  the  fact  that  a  portion  of  the  custard 
was  flavored  with  lemon,  and  the  lemon  cream  did  not 
affect  any  one  unpleasantly.  Fortunately  some  of  the 
vanilla  extract  remained  in  the  bottle  from  which  the  fla- 
voring for  the  ice-cream  had  been  taken,  and  this  was  for- 
warded to  the  chemists.  Each  of  the  experimenters  took 
at  first  thirty  drops  of  the  vanilla  extract,  and  no  ill- effects 
following  this,  one  of  them  took  two  teaspoon fuls  more, 
with  no  results.  This  proved  the  non-poisonous  nature  of 
the  vanilla  more  satisfactorily  than  could  have  been  done 
by  a  chemical  analysis. 

Later,  it  was  found  that  that  portion  of  the  custard 
which  had  been  flavored  with  lemon  was  frozen  immedi- 
ately ;  while  that  portion  which  was  flavored  with  vanilla 
and  which  proved  to  be  poisonous,  was  allowed  to  stand 
for  some  hours  in  a  building,  which  is  described  as  follows 
by  a  resident  of  the  village : 

"  The  cream  was  frozen  in  the  back  end  of  an  old 
wooden  building  on  Main  Street.  It  is  surrounded  by 
shade,  has  no  underpinning,  and  the  sills  have  settled  into 
the  ground.  There  are  no  eve-troughs,  and  all  the  water 
falling  from  the  roof  runs  under  the  building,  the  streets 
on  two  sides  having  been  raised  since  the  construction  of 
the  house.  The  building  had  been  unoccupied  for  a  num- 
ber of  months,  consequently  had  had  no  ventilation,  and 
what  is  worse,  the  back  end  (where  the  cream  was  frozen) 
was  last  used  as  a  meat  market.  The  cream  which  was 
affected  was  that  portion  last  frozen  ;  consequently  it  stood 
in  an  atmosphere  like  that  of  a  privy  vault  for  upward  of 
an  hour  and  a  half  or  two  hours  before  being  frozen." 

The  symptoms  observed  in  these  cases  are  given  by  Dr. 
Mofitt  as  follows : 

"About  two  hours  after  eating  the  cream  every  one  was 
taken  with  severe  vomiting,  and  after  from  one  to  six 
hours  later  with  purging.  The  vomit  was  of  a  soapy  char- 
acter, and  the  stools  watery  and  frothy.  There  was  some 
griping  of  the  stomach  and  abdomen,  with  severe  occipital 
headache,  excruciating  backache,  and  bone  pains  all  over. 


POISONOUS    ICE-CREAM.  81 

especially  marked  iu  the  extremities.  The  vomiting  lasted 
from  two  to  three  hours,  then  gradually  subsided,  and 
everybody  felt  stretchy,  and  yawned  in  spite  of  all  resist- 
ance. The  throats  of  all  were  oedematous.  One  or  two 
were  stupefied ;  others  were  cold  and  experienced  some 
muscular  spasms.  A  numb  feeling,  with  dizziness  and 
momentary  loss  of  consciousness,  was  complained  of  by 
some.  Temperature  was  normal,  and  pulse  from  90  to 
120.  Tongue  dry  and  chapped.  All  were  thirsty  after 
the  vomiting  subsided,  and  called  for  cold  water,  which 
was  allowed  in  small  quantities,  with  no  bad  results. 
After  gettiug  out  no  one  of  the  victims  was  able  to  be  in 
the  hot  sun  for  several  days,  and  even  yet  (about  ten  days 
after  the  poisoning)  the  heat  affects  myself.  I  attended 
twelve  persons,  besides  being  sick  myself,  and  all  were 
affected  in  pretty  much  the  same  way.  Several  complain 
yet  of  inability  to  retain  food  on  the  stomach  without  dis- 
tressing them.  The  man  who  made  the  cream  took  a  tea- 
spoonful  of  it,  and  he  vomited  the  same  as  those  who  took 
a  whole  dish,  but  not  so  often  or  for  so  long  a  time.  All 
are  affected  with  an  irresistible  desire  to  sleep,  which  can 
scarcely  be  overcome.  Even  yet,  some  of  us  feel  that 
drowsy  condition,  with  occasional  occipital  headache." 

The  tyrotoxicon  obtained  from  this  cream  was  adminis- 
tered to  a  kitten  about  two  months  old.  Within  ten 
minutes  the  cat  began  to  retch  and  soon  it  vomited.  This 
retching  and  vomiting  continued  for  two  hours,  during 
which  the  animal  was  under  observation,  and  the  next 
morning  it  was  observed  that  the  animal  had  passed  several 
watery  stools.  After  this,  although  the  kitten  could  walk 
about  the  room,  it  was  unable  to  retain  any  food.  Several 
times  it  was  observed  to  lap  a  little  milk,  but  on  doing  so 
it  would  immediately  begin  to  retch  and  vomit.  Even  cold 
water  produced  this  effect.  This  condition  continuing, 
after  three  days  the  animal  was  placed  under  ether  and  its 
abdominal  organs  examined.  Marked  inflammation  of  the 
stomach  was  supposed  to  be  indicated  by  the  symptoms, 
but  the  examination  revealed  the  stomach  and  small  intes- 
tine filled  with  a  frothy,  serous  fluid,  such  as  had  formed 


82  BACTERIAL    POISONS. 

a  portion  of  the  vomited  matter,  and  the  mucous  membrane 
very  white  and  soft.  There  was  no't  the  slightest  redness 
anywhere.  The  liver  and  other  abdominal  organs  seemed 
normal. 

A  bit  of  the  solid  portion  of  this  cream  was  added  to 
some  normal  milk,  which,  by  the  addition  of  eggs  and 
sugar,  was  made  iuto  a  custard.  The  custard  was  allowed 
to  stand  for  three  hours  in  a  warm  room,  after  which  it 
was  kept  in  an  ice-box  until  submitted  to  chemical  analysis. 
In  this  tyrotoxicon  was  also  found. 

Tyrotoxicon  has  since  been  found  in  some  chocolate 
cream  which  poisoned  persons  at  Geneva,  N.  Y.,  and  in 
lemon  cream  from  Amboy,  Ohio. 

Schearer  reports  the  finding  of  tyrotoxicon  in  both 
vanilla  and  lemon  ice-cream  which  made  many  sick  at 
Nugent,  Iowa. 

Allaben  reports  poisoning  with  lemon  cream,  and 
makes  the  following;  interesting;  statements  concerning  it : 

"  I  would  first  say  July  4,  5,  and  6  were  very  warm. 
Monday  evening,  July  5,  the  custards  were  cooked,  made 
from  Monday  morning's  cream  and  Monday  night's  milk, 
boiled  in  a  tin  pan  that  had  the  bright  tin  worn  off.  It 
was  noticed  that  one  pan  of  cream  was  not  sweet,  but 
thinking  it  would  make  no  difference,  it  was  used;  the 
freezers  were  thoroughly  cleaned  and  scalded,  and  the 
custards  put  in  the  same  evening  while  hot;  the  cream  was 
frozen  Tuesday  afternoon,  having  stood  in  the  freezers 
since  the  night  before,  when  the  weather  was  very  warm." 

No  analysis  of  this  cream  was  made,  but  the  symptoms 
agree  with  those  of  tyrotoxicon  poisoning. 

Weeford  observed  several  cases  of  poisoning  from 
custard  flavored  with  lemon.  These  custards  were  tested 
for  mineral  poisons,  with  negative  results. 

Morrow  has  put  forth  the  claim  that  ice-cream  poison- 
ing is  solely  due  to  artificially  prepared  vanillin,  which  is, 
according  to  his  statement,  used  instead  of  vanilla  extract, 
but  the  facts  stated  above  concerning  poisoniug  with  creams 
in  which  other  flavors  had  been  used  contradict  this  claim. 
Moreover,  Gibson  has  shown  the  utter  absurdity  of  the 


POISONOUS    MEAL    AND    BREAD.  83 

claim,  inasmuch  as  he  calculates  from  the  amount  of  flavor- 
ing ordinarily  used  in  ice-cream,  that  in  order  to  produce 
the  toxic  symptoms  observed,  the  flavoring  must  be  ten 
times  as  poisonous  as  pure  strychnine. 

Bartley  suggests  that  poisonous  cream  sometimes 
results  from  the  use  in  its  manufacture  of  poor  or  putrid 
gelatin.  This  is  highly  probable,  and  with  the  gelatin 
the  germs  of  putrefaction  may  be  added  to  the  milk. 

Poisonous  Meal  and  Bread. — Eeference  has  already 
beeu  made  to  the  fact  that  the  peasants  in  certain  parts  of 
Italy  are  frequently  poisoned  by  eating  mouldy  corn-meal. 
As  has  also  been  stated,  Lombroso  and  others  have  ob- 
tained from  this  meal  ptomaines,  some  of  which  give  the 
same  color  reaction  as  strychnine.  In  1886,  Ladd  suc- 
ceeded in  isolating  from  "  heated  "  corn-meal  a  ptomaine 
which  forms  in  urea-like  crystals.  The  quantity  was  not 
sufficient  for  an  ultimate  analysis,  and  the  physiological 
action  has  not  been  studied.  Poisoning  from  decomposed 
and  mouldy  bread  is  not  unknown. 


CHAPTER  IV. 

GENERAL     CONSIDERATIONS     OF    THE    RELATION    OF 
BACTERIAL    POISONS    TO    INFECTIOUS    DISEASES. 

The  majority  of  diseases  may  be  grouped  from  an  etio- 
logical standpoint  into  the  following  classes:  (1)  Trau- 
matic ;  (2)  infectious ;  (3)  autogenous  ;  and  (4)  neurotic.  It 
must  be  understood,  however,  that  in  many  diseases  the 
cause  is  not  single,  but  multiple,  and  for  this  reason  sharp 
lines  of  classification  cannot  be  drawn.  For  instance,  the 
greatest  danger  in  those  traumatic  affections  in  which  the 
traumatism  itself  does  not  cause  death,  lies  in  infection. 
The  wound  has  simply  provided  a  suitable  point  of  en- 
trance for  the  infecting  agent.  Indeed,  the  break  in  the 
continuity  of  tissue  may  be  so  slight  that  it  is  of  import 
and  danger  only  on  account  of  the  coincident  infection. 
This  is  true  in  many  cases  of  tetanus.  Furthermore,  an 
infectious  disease,  whether  it  originates  in  a  traumatism  or 
not,  is  markedly  influenced  by  what  we  are  pleased  to  call 
the  idiosyncrasy  of  the  patient,  and  by  which  we  mean  the 
peculiarities  of  tissue  metabolism  taking  place  in  the  indi- 
vidual at  the  time.  A  dozen  men  may  be  exposed  alike  to 
the  same  infection,  and  the  infecting  agent  may  find  a  suit- 
able soil  for  its  growth  and  development  in  two  of  these, 
while  in  the  other  ten  this  same  agent  meets  with  such 
adverse  influences  that  it  dies  without  producing  any  appre- 
ciable effects ;  or  all  may  be  infected,  but  with  difference  in 
degree,  as  is  evidenced  by  variation  in  symptoms,  in  the 
length  of  time  that  this  infecting  agent  continues  to  grow 
and  develop  in  the  body  and  in  the  ultimate  result.  Every 
physician  who  has  had  experience  in  the  treatment  of 
typhoid  fever,  diphtheria,  scarlet  fever,  or,  in  short,  of  any 
of  the  infectious  diseases,  will  appreciate  the  importance  of 
the  personal  equation  in  his  patients. 


RELATION    TO    INFECTIOUS    DISEASES.  85 

Chaerin  and  Roger  have  shown  that  white  rats, 
which  are  naturally  immune  to  anthrax,  become  susceptible 
when  fatigued  by  being  kept  on  a  small  tread -mill.  Eleven 
rats  were  inoculated  with  an  anthrax  culture ;  five  of  these 
which  were  allowed  to  rest  in  the  cage  manifested  no  symp- 
toms of  the  disease,  while  six  which  were  placed  on  the 
tread-mill  developed  the  disease  and  died  within  from 
twenty-four  to  thirty  hours.  The  bacilli  were  found  in 
the  liver  and  spleen  of  those  which  died ;  and  guinea-pigs 
inoculated  with  these  germs  died.  The  influence  of  the 
condition  of  health  on  susceptibility  to  the  infectious  dis- 
eases has  also  been  shown  by  Leo,  who  found  that  mice 
which  are  naturally  insusceptible  to  glanders,  become  highly 
susceptible  when  they  are  rendered  diabetic  by  the  adminis- 
tration of  phloridzin. 

That  some  neurotic  affections  originate  from  traumatism 
we  know.  That  others  of  this  class  are  largely  due  to  mal- 
nutrition accompanied  by  improper  metabolism  or  insuffi- 
cient elimination,  or,  in  other  words,  are  to  some  extent 
autogenous,  all  believe.  Understanding,  then,  that  the 
above  classification  does  not  attempt  a  sharp  and  marked 
differentiation  of  the  causes  of  disease,  we  will  now  give 
our  attention  to  a  consideration  of  the  chemical  factors  in 
the  causation  of  the  infectious  diseases,  and  of  the  trau- 
matic, autogenous  and  neurotic,  in  so  far  as  these  are  influ- 
enced by  infection. 

Recognizing  the  fact  that  germs  do  bear  a  causal  relation 
to  some  diseases,  the  question  arises,  How  do  these  organ- 
isms produce  disease?  In  what  way  does  the  bacillus 
anthracis,  for  instance,  induce  the  symptoms  of  the  disease 
and  death?  Many  answers  to  this  question  have  been 
offered.  Some  of  the  most  important  of  these  are  as  fol- 
lows : 

1.  It  was  first  suggested  by  Bollinger  that  apoplecti- 
form anthrax  is  due  to  deoxidation  of  the  blood  by  the 
bacilli.  These  germs  are  aerobic,  and  were  supposed  to 
deprive  the  red  blood-corpuscles  of  their  oxygen.  This 
theory  was  suggested  most  probably  by  the  resemblance  of 
the  symptoms  to  those  of  carbonic  acid  po:soning.     The 


5b  BACTEEIAL    POISONS. 

most  prominent  of  these  symptoms  are  dyspnoea,  cyanosis, 
convulsions,  dilated  pupils,  subnormal  temperature,  and, 
in  general,  the  phenomena  of  asphyxia.  Moreover,  post- 
mortem examination  reveals  conditions  similar  to  those 
observed  after  death  by  deprivation  of  oxygen.  The  veins 
are  distended,  the  blood  is  dark  and  thick,  the  parenchy- 
matous organs  are  cyanotic,  and  the  lungs  hyperaemic. 
Bollinger  compared  this  form  of  anthrax  to  poisoning 
with  hydrocyanic  acid,  which  was  then  believed  to  produce 
fatal  results  by  robbing  the  blood  of  its  .oxygen. 

This  theory  was  supported  by  the  observations  of  SzPiL- 
mann,  who  found  that  while  the  putrefactive  bacteria  are 
destroyed  by  ozone,  the  bacillus  anthracis  thrives  and  mul- 
tiplies in  this  gas. 

This  theory  pre-supposed  a  large  number  of  bacilli  in  the 
blood,  and  this  accorded  with  the  estimate  of  Davaine, 
which  placed  the  number  at  from  eight  to  ten  million  in  a 
single  drop.  But  more  extended  and  careful  observation 
showed  that  the  blood  of  animals  dead  from  anthrax  is 
often  very  poor  in  bacilli.  ViRCHOW  reported  cases  of 
this  kind.  Bollinger  himself  found  the  bacilli  often 
confined  to  certain  organs  and  not  abundant  in  the  blood. 
Then  Siedamgrotzky  counted  the  organisms  in  the  blood 
in  various  cases  and  found  not  only  that  the  estimate  made 
by  Davaine  is  too  large,  but  that  in  many  instances  the 
number  present  in  the  blood  is  small.  Joffroy  found  in 
some  of  his  inoculation  experiments  that  the  animals  died 
before  any  bacilli  appeared  in  the  blood.  These  and  other 
investigations  of  similar  character  began  to  cause  workers 
in  this  field  of  research  to  doubt  the  truth  of  the  theory  of 
Bollinger,  and  these  doubts  were  soon  converted  into 
positive  evidence  against  it.  Pasteur,  in  support  of  the 
theory,  reported  that  birds  were  not  susceptible  to  anthrax, 
and  he  accounted  for  this  by  supposing  that  the  blood 
corpuscles  in  birds  do  not  part  with  their  oxygen  readily. 
However,  it  was  shown  by  Oemler  and  Feser  that  the 
learned  Frenchman  had  generalized  from  limited  data,  and 
that  many  birds  are  especially  susceptible  to  the  disease. 
Oemler  found  that  the  blood  even  when  rich  in  bacilli 


RELATION    TO    INFECTIOUS    DISEASES.  87 

still  possesses  the  bright-red  color  of  oxy-ha?moglobin. 
Toepper  and  Roloff  reported  cases  of  apoplectiform 
anthrax  in  which  there  was  no  difficulty  in  respiration. 
Toussaint  caused  animals  which  had  been  inoculated  with 
the  anthrax  bacillus  to  breathe  air  containing  a  large 
volume  of  oxygen,  and  found  that  this  did  not  modify  the 
symptoms  or  retard  death.  Finally,  Nencki  determined 
the  amount  of  physiological  oxidation  going  on  in  the  bodies 
of  animals  sick  with  anthrax  by  estimating  the  amount  of 
phenol  excreted  after  the  administration  of  one  gramme  of 
benzol,  and  found  that  the  oxidation  of  the  benzol  was  not 
diminished  by  the  disease.  Thus,  the  theory  that  germs 
destroy  life  by  depriving  the  blood  of  its  oxygen  has  been 
found  not  to  be  true  for  anthrax,  and  if  not  true  for 
anthrax,  certainly  it  cannot  be  for  any  other  known  disease. 
The  bacillus  anthracis  is,  as  has  been  stated,  aerobic,  while 
most  of  the  pathogenic  bacteria  are  anaerobic — that  is,  they 
live  in  the  absence  of  oxygen.  This  element  is  not  neces- 
sary to  their  existence,  and,  indeed,  when  present  in  large 
amount,  it  is  fatal  to  them.  Moreover,  in  many  diseases, 
the  bacteria  are  not  found  in  the  blood  at  all.  Lastly,  the 
symptoms  of  these  diseases  are  not  those  of  asphyxia.  These 
facts  have  caused  all  bacteriologists  to  acknowlege  that  this 
theory  is  not  the  right  one. 

2.  If  a  properly  stained  section  of  a  kidney  taken  from 
a  guinea-pig,  which  has  been  inoculated  with  the  bacillus 
anthracis,  be  examined  under  a  microscope,  the  bacilli  will 
be  found  to  be  present  in  such  large  numbers  that  they  form 
einboli,  which  not  only  close,  but  actually  distend  the  capil- 
laries and  larger  bloodvessels,  and  interfere  with  the  normal 
functions  of  the  organ.  A  similar  condition  is  sometimes 
found  on  microscopical  examination  of  the  liver,  spleen, 
and  lungs.  From  these  appearances,  it  was  inferred  by 
Bollinger  that  the  bacilli  produce  the  diseased  condition 
simply  by  accumulating  in  large  numbers  in  these  impor- 
tant organs,  and  mechanically  interrupting  their  functions. 
This  is  known  as  the  mechanical  interference  theory. 

Klebs  and  Toussaint  were  formerly  ardent  advocates 
of  this  theory  in  its  application  to  anthrax,  and  the  latter 


8b  BACTERIAL    POISONS. 

thought  that  the  symptoms  and  death  are  due  to  stoppage 
of  the  pulmonary  circulation  by  means  of  emboli.  How- 
ever, Hoffa  studied  this  point  by  making  numerous  post- 
mortem examinations,  and  was  unable  to  confirm  it.  A 
like  result  followed  the  work  of  Virchow,  Colin,  and 
Siedamorotzky,  and  the  mechanical-interference  theory 
has  been  abandoned. 

In  the  majority  of  germ  diseases  this  theory  never  had 
any  support.  There  is  not  found  any  great  accumulation 
of  bacteria  in  any  organ,  and  the  number  and  distribution 
of  the  germs  are  such  that  the  theory  of  mechanical  inter- 
ference cannot  be  held. 

3.  Another  answer  given  to  the  question,  How  do  germs 
cause  disease?  is,  that  they  do  so  by  consuming  the  proteids 
of  the  body  and  thus  deprive  it  of  its  sustenance.  The 
proteids  are  known  to  be  necessary  for  the  building  up  of 
cells,  and  it  is  also  known  that  microorganisms  feed  upon 
proteids.  But  this  theory  is  untenable  for  several  reasons. 
In  the  first  place,  many  of  the  infectious  diseases  destroy 
life  so  quickly  that  the  fatal  effect  cannot  be  supposed  to 
be  due  to  the  consumption  of  any  very  large  amount  of 
proteids.  In  the  second  place,  the  distribution  of  the  micro- 
organisms is  such  in  many  diseases  that  they  do  not  come 
in  contact  with  any  large  proportion  of  the  proteids  of  the 
body.  In  the  third  place,  the  symptoms  of  the  majority  of 
these  diseases  are  not  those  which  would  be  produced  by 
withdrawing  from  the  various  organs  their  food.  The 
symptoms  are  not  those  of  general  starvation. 

4.  Still  another  theory,  which  has  been  offered,  is  that 
the  bacteria  destroy  the  blood  corpuscles,  or  lead  to  their 
rapid  disintegration.  But  in  many  of  the  infectious  dis- 
eases, as  has  been  stated,  the  microorganisms,  although  very 
abundant  in  some  organs,  are  not  present  in  the  blood. 
Moreover,  the  disintegration  of  the  blood  corpuscles  is  not 
confirmed  by  microscopical  examination. 

5.  Seeing  the  vital  deficiencies  in  the  above  theories,  and 
being  impressed  by  the  results  obtained  by  the  chemical 
study  of  putrefaction,  bacteriologists  have  been  led  to  in- 
quire into  the  possibility  of  the  symptoms  of  the  infectious 


RELATION"    TO    INFECTIOUS    DISEASES.  89 

diseases  being  due  to  chemical  poisons.     In  investigating 
this  theory,  three  possibilities  suggest  themselves  : 

(a)  The  microorganisms  themselves  may  be  poisonous, 
or  the  poison  may  be  an  integral  part  of  them.  Neelsen, 
at  one  time  an  advocate  of  this  theory,  thus  accounted  for 
the  appearance  and  increase  in  violence  of  the  symptoms  as 
the  germs  increase  in  number.  In  order  for  the  conditions 
of  this  theory  to  be  fulfilled  the  microorganisms  must  be 
present  in  the  blood  before  any  of  the  symptoms  appear. 
But  in  anthrax  the  most  thoroughly  studied  of  all  the  in- 
fectious diseases,  and  the  one  to  which  all  these  theories 
have  been  applied,  the  bacilli  first  appear  in  the  blood,  as  a 
rule,  only  a  few  hours  before  death,  and  long  after  the 
appearance  of  the  first  symptoms ;  while  in  many  other 
diseases  the  germs  are  never  found  in  the  blood.  More- 
over, as  Hoffa  has  shown,  if  this  theory  be  true,  the  in- 
jection of  a  large  quantity  of  anthrax  bacilli  directly  into 
the  blood  should  be  followed  immediately  by  symptoms  of 
the  disease,  and  death  should  be  speedy.  But  he  found,  on 
making  experiments  of  this  kind,  that  the  symptoms  did 
not  appear  until  from  twenty-four  to  seventy-two  hours. 
Nencki  found  by  analysis  that  the  substance  of  the  an- 
thrax bacilli  resembles  vegetable  casein  in  some  respects, 
and  animal  mucin  in  others.  This  "  anthrax  protein "  is 
freely  soluble  in  alkalies,  is  insoluble  in  water,  acetic  acid, 
and  the  dilute  mineral  acids.  It  contains  no  sulphur  and 
was  believed  by  Nencki  to  be  inert ;  but  the  recent  re- 
searches of  Buchner  has  shown  that  this  belief  is  not  well 
founded.  It  has  been  stated  by  a  number  of  investigators 
that  suppuration  might  be  induced  by  the  injection  of  cer- 
tain sterilized  cultures,  but  the  dictum  of  Weigert,  "  no 
suppuration  without  bacteria,"  has  been  generally  accepted  ; 
and  statements  to  the  contrary,  although  some  of  them  have 
been  made  by  men  of  excellent  reputation,  have  until  recently 
received  but  little  credence.  However,  Buchner  has  shown 
conclusively  that  the  albuminate  of  the  bacterial  cell  in  as 
many  as  seventeen  different  species  possesses  well-marked 
pyogenetic  properties,  and  that  the  pus  formed  is  free  from 
germs.     Buchner  separated  the  microorganisms  from  the 


90  BACTERIAL    POISONS. 

soluble  substances  accompanying  them  by  sedimentation  and 
decantation,  washed  the  cells,  dissolved  them  in  a  0.5  per 
cent,  solution  of  potash  by  the  aid  of  heat,  precipitated  the 
albumin  with  dilute  mineral  acid,  and,  after  repeated  re- 
solution in  alkali  and  reprecipitation  with  acid,  employed 
the  purified  proteid  in  his  experiments.  Introduced  with 
antiseptic  precautions  under  the  skin,  this  substance  invari- 
ably causes  suppuration.  This  demonstrates  that  the  sub- 
stance of  the  bacterial  cell  is  not  altogether  inert.  It  is 
impossible  at  present  to  say  to  what  extent  the  course  of 
an  infectious  disease  may  be  influenced  by  the  breaking 
down  of  a  large  number  of  bacterial  cells  and  the  intro- 
duction of  their  substance  into  the  blood. 

(6)  The  microorganisms  may  be  intimately  associated 
with  or  may  produce  a  soluble,  chemical  ferment,  which, 
by  its  action  on  the  body,  produces  the  symptoms  of  the ' 
disease  and  death.  This  theory  formerly  had  a  number  of 
ardent  supporters,  among  whom  might  be  mentioned  the 
eminent  scientist,  de  Bary.  But  Pasteur  proved  the 
theory  false  when  he  filtered  anthrax  blood  through  earthen 
cylinders,  inoculated  animals  with  the  filtrate,  and  failed  to 
produce  any  effect.  Nenckt  made  a  similar  demonstration 
when  he  inoculated  a  two  per  cent,  gelatin  preparation 
with  the  anthrax  bacillus,  which  liquefied  the  p reparation, 
and  on  standing  the  bacilli  settled  to  the  bottom.  The 
supernatant  fluid,  which  was  clear,  alkaline  in  reaction,  and 
contained  dissolved  "  anthraxprotein,"  was  filtered  and 
injected  into  animals  without  producing  any  effect.1 

It  must  not  be  inferred  from  the  above  statements  that 
bacteria  do  not  produce  any  ferments.  Many  of  them  do 
form  both  diastatic  and  peptic  ferments,  which  may  retain 
their  activity  after  the  bacteria  have  been  destroyed ;  but 
there  is  no  proof  that  in  any  case  these  ferments  have  any 
causal  relation  to  the  disease.  After  the  diseased  process 
has  been  inaugurated  some  of  these  ferments  probably  play 

1  We  now  know  that  if  the  supernatant  fluid  used  in  this  experiment 
had  been  injected  in  sufficient  quantity  death  would  have  been  produced 
by  the  soluble  chemical  poisons. 


RELATION    TO    INFECTIOUS    DISEASES.  91 

an  important  part  in  the  production  of  morphological 
changes,  the  nature  of  which  will  be  indicated  when  these 
diseases  are  discussed. 

(c)  The  germ  may  produce  chemical  poisons  by  splitting 
up  preexisting  complex  compounds  in  the  body.  This 
theory  finds,  in  the  first  j)lace,  strong  support  in  the  well- 
known  fact  that  many  of  the  putrefactive  germs  produce 
highly  poisonous  bodies ;  and,  in  the  second  place,  the  for- 
mation of  chemical  poisons  will  account  for  the  appearance 
of  the  symptoms  of  the  disease  when  the  microorganisms 
never  find  their  way  into  the  blood.  The  correctness  of 
this  theory  has  been  tested  by  a  large  number  of  investi- 
gators, and  with  the  result  that  its  truth  has  been  firmly 
established.  It  was  soon  found  that  pathogenic  germs 
grown  in  meat  broth  and  other  culture  media  elaborate 
chemical  poisons  which,  when  injected  into  the  lower  animals, 
induce  in  an  acute  form  one  or  more  of  the  symptoms  char- 
acteristic of  the  disease  caused  in  man  by  the  microorgan- 
ism. It  is  true  that  until  quite  recently  this  theory  has 
been  opposed  by  some,  and  it  is  altogether  possible  that 
at  present  there  may  be  those  who  are  not  altogether  con- 
vinced of  its  truth.  However,  we  are  not  acquainted  with 
any  argument  against  it  which  remains  unanswered.  For 
a  while  Baumgarten  claimed  that  the  formation  of  chem- 
ical poisons  in  the  dead  matter  of  meat  broth  and  other 
media  by  the  germ  does  not  prove  that  the  same  agent  is 
capable  of  forming  the  same  or  similar  products  within  the 
living  body  ;  but  the  isolation  of  tetanine  from  the  ampu- 
tated arm  of  a  man  with  tetanus,  by  Brieger,  furnished 
the  first  positive  answer  to  this  criticism,  and  since  that 
time  a  number  of  bacterial  poisons  have  been  obtained  from 
the  bodies  of  men  and  the  lower  animals.  We  now  expect 
to  find  each  specific,  pathogenic  microorganism  producing 
its  characteristic  poison  or  poisons.  The  evidence  on 
this  point  will  be  given  further  on  in  a  brief  sketch  of  the 
chemical  factors  in  the  causation  of  some  of  the  best-known 
infectious  diseases. 

Before  taking  up  the  individual  diseases,  we  will  give 


92  BACTERIAL    POISONS. 

what  appears  to  us,  in  the  present  state  of  our  knowledge, 
a  correct  definition  of  an  infectious  disease. 

An  infectious  disease  arises  when  a  specific,  pathogenic 
microorganism,  having  gained  admittance  to  the  body,  and 
having  found  the  conditions  favorable,  grows  and  multi- 
plies, and  in  so  doing  elaborates  a  chemical  poison  which 
induces  its  characteristic  effects. 

In  the  systemic  infectious  diseases,  such  as  anthrax, 
typhoid  fever,  and  cholera,  this  poison  is  undoubtedly  taken 
into  the  general  circulation,  and  affects  the  central  nervous 
system.  In  the  local  infectious  diseases,  such  as  gonorrhoea, 
and  infectious  ophthalmia,  the  principal  action  of  the  poison 
seems  to  be  confined  to  the  place  of  its  formation.  Though 
even  in  these,  when  of  a  specially  virulent  type,  the  effects 
may  extend  to  the  general  health.  It  may  be  that  in  some 
diseases  the  chemical  poison  has  both  a  local  and  a  systemic 
effect.  Thus,  it  is  by  no  means  certain  that  the  ulceration 
of  typhoid  fever  is  due  directly  to  the  bacillus.  On  the 
other  hand,  it  is  altogether  probable  that  the  anatomical 
changes  in  the  intestine  result  from  the  irritating  effects  of 
the  poison  at  the  place  of  its  formation. 

With  the  proof,  that  the  deleterious  effects  wrought  by 
germs  are  due  to  chemical  poisons  elaborated  by  them 
during  their  growth,  admitted,  let  us  inquire  what  proper- 
ties a  microorganism  must  possess  before  it  can  be  said  to 
be  the  specific  cause  of  a  disease.  The  four  rules  of  Koch 
have  been  generally  conceded  to  be  sufficient  to  show  that 
a  given  germ  is  the  sole  and  sufficient  cause  of  the  disease 
with  which  that  germ  is  associated.  Briefly,  these  rules  are 
as  follows  : 

1 .  The  germ  must  be  present  in  all  cases  of  that  disease. 

2.  The  germ  must  be  isolated  from  other  organisms 
and  from  all  other  matter  found  with  it  in  the  diseased 
animal. 

3.  The  germ  thus  freed  from  all  foreign  matter  must, 
when  properly  introduced,  produce  the  disease  in  healthy 
animals. 

4.  The    microorganism    must    be    found   properly  dis- 


KELATION    TO    INFECTIOUS    DISEASES.  93 

tributed  in  the  animal  in  which  the  disease  has  been 
induced. 

Let  us  give  our  special  attention  to  the  first  of  these 
rules  for  a  few  moments.  What  is  meant  by  the  state- 
ment that  the  special  germ  must  be  found  in  every  case  of 
the  disease?  How  will  A,  pursuing  his  studies  on  the 
bacteriology  of  a  given  disease  in  America,  decide  whether 
or  not  a  bacillus  which  he  finds  is  identical  with  one  which 
has  been  reported  as  invariably  present  in  the  same  disease 
by  B,  who  has  investigated  an  epidemic  in  Germany  ? 
What  means  are  relied  upon  to  prove  the  identity  of  these 
two  organisms  ?  The  means  which  have  been  relied  upon 
wholly  are  the  form,  size,  reaction  with  staining  reagents, 
manner  of  growth  on  various  nutrient  media,  and,  in  ex- 
ceptional instances,  correspondence  in  their  effects  upon  the 
lower  animals.  In  other  words,  with  the  exception  of  those 
instances  in  which  the  effects  upon  animals  are  tried,  the 
characteristic  property  by  which  the  germ  causes  the  disease 
is  left  wholly  out  of  consideration.  It  is  admitted  that  any 
causal  relation  which  the  germ  may  have  to  the  disease  is 
due  to  its  capability  of  forming  one  or  more  chemical  poi- 
sons, and  yet  no  attempt  is  made  to  ascertain  whether  or 
not  it  possesses  this  property.  Indeed,  some  of  the  most 
eminent  bacteriologists  have  taught  that  in  the  identifica- 
tion of  germs  the  reactions  with  staining  reagents  and  the 
appearance  of  the  growths  on  the  various  nutritive  media 
are  of  more  importance  than  the  observation  of  the  effects 
upon  animals.     Thus,  Flugge  says  : 

"  Inoculation  experiments  with  both  typhoid  dejections 
and  pure  cultures  of  the  Eberth  bacillus  have  universally 
been  without  success.  The  few  experiments  in  which  a 
typhoid  disease  has  followed  inoculation  or  feeding  have 
been  made  with  impure  material  containing  other  active 
bacteria.  It  is  known  that  a  group  of  widely  distributed 
organisms,  which,  however,  are  wholly  different  from  the 
typhoid  bacillus,  have  the  power,  when  injected  subcu- 
taneously  or  intravenously,  of  producing  in  animals  death 
with  marked  swelling  and  ulceration  of  Peyer's  patches. 
To  these  organisms  undoubtedly  are  due  the  apparently 

5* 


94  BACTERIAL    POISONS. 

positive  results  which  some  authors  have  supposed  to  be 
due  to  inoculation  with  the  typhoid  bacillus." 

In  other  words,  this  eminent  author  teaches  that  although 
other  germs  may  cause  the  essential  symptoms  and  lesions 
of  typhoid  fever  in  the  lower  animals,  they  are  not  related 
to  the  germ  found  in  the  spleen  of  man  after  death  from 
typhoid  fever,  because  they  do  not  react  in  the  same  man- 
ner with  the  auilin  stains,  and  present  a  different  appear- 
ance in  their  growths  on  potatoes. 

We  will  suppose  that  in  an  epidemic  of  diphtheria,  A 
examines  the  membrane  from  a  hundred,  or  we  might  as 
well  suppose  a  thousand,  children,  and  finds  a  characteristic, 
well-marked,  easily  recognized  bacillus  in  all.  He  isolates 
this  organism,  and  obtains  it  in  pure  culture.  He  inocu- 
lates animals,  and  these  manifest  all  the  signs,  together  with 
the  appearance  of  the  characteristic  membrane  of  diphtheria, 
and  in  these  animals  he  finds  his  bacillus  growing  as  in  the 
throats  of  the  children.  All  the  rules  of  Koch  have  been 
complied  with.  Has  A  demonstrated  that  his  bacillus  is  the 
sole  cause  of  diphtheria  ?  No.  He  has  shown  that  his 
bacillus  is  a  cause  of  diphtheria ;  but  he  has  not  proven 
that  there  may  not  be  other  germs,  wholly  different  from 
his  in  form  and  size,  which  may  also  cause  diphtheria. 
The  most  which  can  be  proven  by  Koch's  rules  is  that  a 
given  germ  is  a  cause  of  a  certain  disease.  They  do  not 
show,  as  most  bacteriologists  would  have  us  believe,  that 
the  given  germ  is  the  sole  cause  of  the  disease. 

To  illustrate,  we  will  suppose  that  a  botanist  in  visiting 
Arabia  should  find  a  tree  producing  a  berry,  the  coffee 
berry,  which,  when  properly  prepared  and  taken  into  the 
system,  produces  certain  effects  which  are  due  to  the  alka- 
loid, caffein,  and  which  invariably  follow  the  drinking  of 
a  decoction  of  these  berries ;  would  our  supposed  discoverer 
be  justified  in  concluding  that  the  coffee  tree  is  the  only 
plant  in  the  world  capable  of  producing  these  supposed 
characteristic  effects  ?  Should  he  reach  such  a  conclusion, 
the  fact  that  it  is  not  warranted  would  be  shown  by  a  study 
of  the  tea  plant  of  China  and  the  guarana  of  South  America. 

The  moment  that  it  is  granted  that  the  real  poison  of  the 


RELATION    TO    INFECTIOUS    DISEASES.  95 

disease  is  chemical  in  character,  it  becomes  evident  that  no 
one  is  justified  in  saying  that  one  germ  is  the  sole  source  of 
that  poison.  Such  a  statement  would  be  as  unwarranted 
as  one  that  the  coffee  tree  is  the  sole  source  of  caffein,  or 
that  the  strychnos  Ignatii  is  the  only  species  of  the  nat- 
ural order  Loganiacese  which  produces  a  convulsive  poison. 
In  other  words,  the  specific  cause  of  a  given  disease  is  not 
to  be  determined  wholly  by  the  morphology  of  the  germ, 
but  by  the  character  of  the  chemical  poison  which  is  the  true 
materies  morbi. 

Bacteria  cannot  be  classified,  so  far  as  their  causal  rela- 
tionship to  disease  is  concerned  (and  this  is  the  most  im- 
portant knowledge  to  be  gained  from  them),  until  we  know 
the  nature  of  their  chemical  products,  for  it  is  by  virtue  of 
these  that  the  germs  have  any  causal  relationship  to  dis- 
ease. 

It  is  possible  that  two  germs  may  be  unlike  in  form,  and 
yet  they  may  produce  poisons  which  are  identical  or  those 
which  are  very  similar  in  their  effects  upon  man.  One 
germ  may  be  stained  by  Gram's  method  and  another  fail 
to  be  acted  upon  when  so  treated ;  but  this  does  not  prove 
that  their  chemical  products  are  totally  unlike.  This  is 
not  only  a  possibility,  it  is  a  fact  which  has  been  demon- 
strated repeatedly,  both  with  pathogenic  and  non-pathogenic 
organisms.  A  few  illustrations  may  be  given  here  :  The 
yeast  plant  is  not  the  only  microorganism  which  will  pro- 
duce alcohol  in  saccharine  solutions.  The  same  product 
results  from  the  growth  of  the  bacterium  Bischleri,  bac- 
terium coli  commune,  bacterium  ilei,  bacterium  ovale  ilei, 
bacterium  lactis  aerogenes,  and  others  (Nencki).  Mor- 
phologically, there  are  marked  differences  between  the  yeast 
plant  and  these  bacteria,  but  they  alike  produce  alcohol. 
More  than  a  dozen  germs,  including  both  micrococci  and 
bacilli,  are  capable  of  generating  lactic  acid.  Some  of  these 
produce  an  acid  which  is  optically  inactive ;  others,  one 
which  is  dextro-rotatory ;  and  others  still,  one  which  is  lsevo- 
rotatory.  The  tetanus  germ  of  Kitasato  and  that  of 
Tizzoni  and  Cantani  are  known  to  be  different.  Cultures 
of  the  former  in  bouillon  are  virulent,  while  those  of  the 


96  BACTEEIAL    POISONS. 

latter  in  the  same  medium  are  inert.  Not  only  are  these 
two  organisms  morphologically  and  biologically  distinct, 
but  their  poisons  are  chemically  unlike.  Brieger  and 
Frankel  precipitated  the  poisonous  albumin  of  the  germ 
of  Kitasato  with  alcohol,  but  this  reagent  renders  the 
poison  of  the  Italian  germ  inert.  Notwithstanding  this 
difference,  however,  both  microorganisms  and  their  chemical 
products  produce  tetanic  convulsions  and  death  in  the  lower 
animals.  We  must,  therefore,  admit  that  there  are  at  least 
two  distinct  germs,  each  of  which  is  capable  of  causing 
tetanus ;  and  how  many  other  bacteria  with  like  properties 
there  may  be  no  one  can  tell.  All  attempts  to  find  a  mor- 
phologically specific  germ  in  the  summer  diarrhoeas  of 
infancy  have  failed.  The  labors  of  Booker  in  this  coun- 
try and  of  Escherich  in  Germany  have  shown  that  no 
one  species  or  variety  is  constantly  present.  No  less  than 
thirty  distinct  germs  have  been  obtained  from  the  bowels 
and  feces  of  children  suffering  from  these  diarrhoeas.  A 
germ  which  is  frequently  present  one  season  may  not  be 
found  at  all  the  next.  Are  we  to  conclude  from  this  fail- 
ure to  comply  with  the  first  of  Koch's  rules,  that  the  sum- 
mer diarrhoeas  of  infancy  are  not  due  to  microorganisms  ? 
Certainly  not ;  especially  in  view  of  the  fact  that  Baginsky 
and  Stadthagen  have  obtained  from  pure  cultures  of  a 
saprophytic  germ  found  in  the  stools  of  cholera  infantum  a 
poisonous  base  and  a  poisonous  proteid;  and  Vaughan 
has  shown  that  at  least  three  of  Booker's  bacteria  pro- 
duce chemical  poisons  which  cause  in  kittens  retching, 
vomiting,  purging,  collapse,  and  death.  To  the  contrary 
we  are  justified  in  concluding  that  these  diarrhoeas  may  be 
due  to  any  one  or  more  of  a  number  of  germs  which  differ 
from  one  another  sufficiently  morphologically  to  be  classified 
as  distinct  species.  The  similarity  among  these  bacteria 
will  not  be  discovered  by  a  study  of  their  size,  form,  and 
reactions  with  staining  agents,  but  by  a  study  of  their 
chemical  products,  the  agents  by  virtue  of  which  they  cause 
the  disease. 

We  think  that  we  are  justified  in  concluding  that  in 
those  diseases  in  which  the  four  rules  of  Koch  have  been 


RELATION    TO    INFECTIOUS    DISEASES.  97 

complied  with,  the  germ  is  a  cause  of  the  disease,  but  our 
range  of  observation  must  be  much  wider  than  it  now  is 
before  we  can  say  that  the  given  germ  is  the  only  cause  of 
the  disease. 

We  believe  that  those  few  infectious  diseases,  such  as 
anthrax  and  tuberculosis,  which  have  such  well-marked, 
typical,  clinical  histories,  are  due  to  equally  well-marked 
and  morphologically  distinct  microorganisms  which  can  be 
recognized  by  microscopical  study  alone ;  but  we  do  not 
believe  that  this  is  true  in  diseases  showing  such  wide  vari- 
ations in  symptoms  as  is  the  case  in  typhoid  fever  and 
cholera  infantum. 

In  all  cases,  we  insist  that  the  true  test  of  the  specific 
character  of  a  germ  is  to  be  made  with  its  chemical  pro- 
ducts. A  given  bacterium  may  not  multiply  in  the  circu- 
lating blood  of  a  dog,  and  failure  to  do  so  is  by  no  means 
proof  that  the  same  organism  might  not  cause  disease  in 
man ;  but  every  germ  which  causes  disease  in  man  does  so 
by  virtue  of  its  chemical  products,  and  if  these  be  isolated 
and  injected  into  the  dog  in  sufficient  quantity  a  poisonous 
effect  will  be  produced.  In  the  study  of  the  bacteriology 
of  the  infectious  diseases,  the  third  and  fourth  of  Koch's 
rules  have  not  been  complied  with  in  many  diseases  on 
account  of  the  insusceptibility  of  the  lower  animals.  The 
majority  of  investigators,  meeting  with  this  difficulty,  have 
been  inclined  to  rest  content  with  the  first  two  rules,  and 
to  conclude  that  when  a  given  germ  is  constantly  present 
in  a  given  disease,  and  not  found  in  other  diseases,  that  it 
is  the  cause  of  the  disease  with  which  it  is  associated.  In- 
deed, we  find  so  good  an  authority  as  Welch  stating  that 
the  successful  inoculation  of  animals  is  not  necessary  in 
order  to  prove  the  causal  relationship  of  a  germ  to  a  disease. 
In  1889,  Vaughan  suggested  that  in  those  instances  in 
which  the  third  and  fourth  of  Koch's  rules  cannot  be 
complied  with  on  account  of  the  insusceptibility  of  the 
lower  animals,  it  must  be  shown  that  the  germ  can  pro- 
duce chemical  poisons  which  will  induce  in  the  lower  ani- 
mals in  an  acute  form  the  characteristic  symptoms  of  the 


98  BACTERIAL    POISONS. 

disease,  before  the  proof  that  the  given  germ  is  the  cause 
of  the  disease  be  accepted  as  positive. 

Heretofore,  the  science  of  bacteriology  has  been  largely 
founded  upon  morphological  .studies.  Bacteriologists  have 
given  their  time  and  attention  to  the  discovery  of  bacterial 
forms  in  the  diseased  organism  and  to  observations  of  char- 
acteristics in  structure  and  growth  of  different  species  of 
bacterial  life.  We  must  now  study  the  physiology  and 
chemistry  of  the  germs,  and  until  this  is  done  we  must 
remain  ignorant  of  the  true  cause  of  disease,  and  so  long 
as  we  remain  ignorant  of  the  cause,  it  cannot  be  expected 
that  we  shall  discover  scientific  and  successful  methods  of 
treatment.  Suppose  that  our  knowledge  of  the  yeast  plant 
was  limited  to  its  form  and  method  of  growth  ;  of  how  little 
practical  importance  this  knowledge  would  be.  That  the 
yeast  plant  requires  a  saccharine  soil  before  it  can  grow, 
that  given  such  a  soil  it  produces  carbonic  acid  gas  and 
alcohol,  are  the  most  important  and  practical  facts  which 
have  been  ascertained  in  its  study.  Likewise,  the  condi- 
tions under  which  pathogenic  germs' multiply  and  the  pro- 
ducts which  they  elaborate  in  their  multiplifi cation  must 
be  ascertained  before  their  true  relationship  to  disease  can 
be  understood. 

In  saying  that  the  moqmological  work  upon  which  the 
science  of  bacteriology  rests  almost  wholly  is  inadequate, 
we  wish  that  it  may  be  plainly  understood  that  we  are  not 
offering  any  hostile  criticism  upon  the  great  men  who  have 
done  this  work  and  who  have  formulated  conclusions  there- 
from. The  development  of  bacteriology  has  been  in  accord- 
ance with  the  natural  law  governing  thfe  growth  of  all  the 
biological  sciences.  The  study  of  form  naturally  and  neces- 
sarily precedes  the  study  of  function.  The  ornithologist 
finds  a  new  species  of  bird.  He  first  studies  its  shape  and 
size,  the  color  of  its  plumage,  the  form  of  its  beak,  the 
number  and  arrangement  of  the  feathers  of  the  tail  and 
wing,  the  color  of  the  eyes,  etc.  All  this  he  can  do  with  a 
single  specimen,  recognizing  the  fact,  however,  that  varia- 
tions more  or  less  marked  are  likely  to  be  found  in  other 
individuals.     More  time  and  wider  opportunities  of  ob- 


KELATION    TO    INFECTIOUS    DISEASES.  99 

servation  will  be  needed  before  he  can  tell  where  and  when 
this  bird  is  accustomed  to  build  its  nest,  upon  what  insects, 
grains,  and  berries  it  feeds,  with  what  other  species  of  birds 
it  lives  iu  peace  and  with  what  it  is  at  war.  A  much 
greater  range  of  observation  and  study  is  necessary  before 
the  naturalist  can  tell  how  his  newly  discovered  species 
would  thrive  if  carried  to  a  new  climate,  where  it  would 
be  compelled  to  live  upon  unaccustomed  food,  to  build  its 
nest  of  strange  material,  and  to  encounter  new  foes. 

We  repeat  that  it  is  no  discredit  to  the  science  nor  to  the 
men  who  have  developed  it  to  say  that  the  study  of  bac- 
teriology has  hitherto  been  almost  wholly  morphological. 
Without  the  morphologist  the  physiologist  and  the  physio- 
logical chemist  could  not  exist.  The  science  having  had 
for  its  support  only  morphological  studies,  the  deductions 
and  formulated  statements  arrived  at  by  its  students,  have 
been  reached  in  accordance  with  the  knowledge  obtained 
from  this  source.  But  now,  it  being  admitted  that  the 
causal  relation  between  a  given  germ  and  a  certain  disease 
is  dependent  upon  the  chemical  products  of  the  groAvth  of 
the  germ,  the  fundamental  lines  of  work  must  be  altered  in 
order  to  correspond  with  this  new  knowledge. 

The  study  of  the  chemical  factors  in  the  causation  of  the 
infectious  diseases  opens  up  for  us  a  field  in  which  much 
work  must  be  done.  Let  us  attempt  a  statement  of  the 
nature  of  some  of  the  researches  that  must  be  carried  out 
along  this  line. 

In  the  first  place,  we  must  ascertain  what  germs  are  toxi- 
cogenic.  This  would  necessitate  a  chemical  study  of  all  kinds 
of  bacteria,  both  the  pathogenic  and  the  non-pathogenic. 
Every  fact  ascertained  in  this  investigation  will  not  have 
its  practical  application  in  medicine,  but  will  have  its 
scientific  value,  and  many  will  most  probably  be  of  more 
or  less  direct  service  to  man. 

Secondly,  it  must  be  determined  under  what  conditions 
these  germs  are  toxicogenic.  It  is  not  at  all  probable  that  all 
those  bacteria  which  are  capable  of  producing  poisons  when 
grown  on  dead  material  outside  of  the  body  are  also  capable 
of  multiplication  and  the  production  of  the  same  substances 


100  BACTERIAL    POISONS. 

when  under  the  influence  of  the  various  secretions  of  the 
body.  Some  bacteria  are  destroyed  by  a  normal  gastric 
juice  within  a  short  time,  while  others  are  not.  The  con- 
ditions of  life  and  growth  are  different  when  the  infecting 
agent  is  introduced  into  the  blood  from  what  they  are  when 
infection  occurs  by  the  way  of  the  alimentary  canal.  This 
is  well  recognized  in  the  two  forms  of  anthrax,  one  of  which 
arises  from  inoculation  through  a  wound  and  the  other  by 
way  of  the  intestines.  A  preventive  treatment  which  is 
efficient  in  one  is  of  no  service  in  the  other.  Then,  again, 
Ave  are  to  study  those  conditions  of  the  blood  and  other 
fluids  of  the  body  which  are  especially  unfavorable  to  the 
successful  implantation  or  the  continued  existence  of  an 
infectious  disease. 

Thirdly,  the  chemical  properties  and  the  physiological 
action  of  these  poisons  will  demand  careful  attention.  Some 
are  especially  depressing  in  their  action  upon  the  heart, 
others  seem  to  manifest  their  chief  energy  upon  the  central 
nervous  system,  while  others  still  act  like  true  gastro- 
intestinal irritants.  In  the  study  of  the  toxicological  effects 
of  these  bacterial  poisons  every  method  of  investigation 
known  in  the  most  advanced  physiological  work  must  be 
employed.  The  action  of  these  agents  on  the  heart,  the 
brain,  the  spinal  cord,  etc.,  must  be  thoroughly  studied. 


CHAPTER  V. 

THE    BACTERIAL    POISONS   OF    SOME    OP    THE    INFECTIOUS 
DISEASES. 

We  will  now  give  our  attention  to  the  chemical  poisons, 
both  the  ptomaines  and  the  proteids,  of  some  of  the  infec- 
tious diseases,  and  in  doing  this  we  will  illustrate  and  sub- 
stantiate the  statements  made  in  the  preceding  chapter. 

Anthrax. — The  definition  of  an  infectious  disease,  as 
we  have  given  it,  is  well  illustrated  by  the  facts  which  have 
been  learned  concerning  the  causation  of  anthrax,  which 
has  probably  been  more  thoroughly  studied  than  any  other 
infectious  disease.  Kausch  taught  that  this  disease  has  its 
origin  in  paralysis  of  the  nerves  of  respiration.  As  to  the 
cause  of  this  paralysis  he  gave  us  no  information.  Delafond 
thought  that  anthrax  has  its  origin  in  the  influence  of  the 
chemical  composition  of  the  soil  affecting  the  food  of  ani- 
mals and  leading  to  abnormal  nutrition.  The  investigations 
of  Gerlach  in  1845  demonstrated  the  contagious  nature 
of  the  disease,  which  was  emphasized  by  Heusinger  in 
1850  and  accepted  by  Virchow  in  1855.  However,  as 
early  as  1849,  Pollender  found  numerous  rod-like  micro- 
organisms in  the  blood  of  animals  with  the  disease.  This 
observation  was  confirmed  by  Brauell,  who  produced 
the  disease  in  healthy  animals  by  inoculations  with  matter 
taken  from  a  pustule  on  a  sick  horse.  Attempts  were  made 
to  ridicule  the  idea  that  these  germs  might  be  the  cause  of 
the  disease,  and  it  was  said  that  the  bodies  seen  were  only 
fine  shreds  of  fibrin  or  blood  crystals.  Some  claimed  that 
the  rod-like  organisms  reported  were  due  to  defects  in  the 
glass,  while  others  claimed  that  the  defects  existed  in  the 
eye  of  the  observer,  and  others  still  suggested  that  the  de- 


102  BACTERIAL    POISONS. 

fects  might  be  found  back  of  the  eye  and  in  the  brain.  But 
in  1863,  Davaine  showed  that  these  little  bodies  must 
have  some  causal  relation  to  the  disease,  inasmuch  as  his 
experiments  proved  that  inoculation  of  healthy  animals 
with  the  blood  of  those  sick  with  anthrax  produced  the 
disease  only  when  taken  at  a  time  when  the  blood  con- 
tained these  organisms.  He  also  demonstrated  beyond  any 
question  that  these  rod-like  bodies  are  bacteria,  capable  of 
growth  and  multiplication.  The  conclusions  of  this  investi- 
gator were  combated  by  many ;  but  Pasteur,  Koch, 
Bollinger,  de  Barry,  and  others,  studied  the  morph- 
ology and  life-history  of  these  organisms,  and  then  came 
the  brilliant  results  of  Pasteur  and  Koch  in  securing 
protection  against  inoculation  anthrax  by  the  vaccination 
of  healthy  animals  with  the  modified  germ  and  subsequent 
inoculation  with  the  virulent  form.  Now,  the  bacillus 
anthracis  is  known  in  every  bacteriological  laboratory,  and 
by  inoculation  with  it  the  disease  is  communicated  at  will 
to  susceptible  animals.  But  here  the  question  arose,  How 
do  these  bacilli  produce  anthrax?  and  in  answer  to  this 
question  the  various  theories  which  we  have  mentioned 
were  proposed. 

The  first  successful  attempt  to  study  the  chemical  poisons 
of  anthrax  was  made  by  Hoffa,  who  obtained  from  pure 
cultures  of  the  bacillus  small  quantities  of  a  ptomaine, 
which,  when  injected  under  the  skin  of  animals,  produces 
the  symptoms  of  the  disease  and  death.  This  substance 
causes  at  first  increased  respiration  and  action  of  the  heart, 
then  the  respirations  become  deep,  slow,  and  irregular ; 
the  temperature  falls  below  the  normal ;  the  pupils  are 
dilated,  and  a  bloody  diarrhoea  sets  in.  On  section  the 
heart  is  found  contracted,  the  blood  dark,  and  ecchymoses 
are  observed  on  the  pericardium  and  peritoneum.  Hoffa 
names  his  poison  anthracin.  Recently  Hoffa  has  isolated 
this  poison  from  the  bodies  of  animals  dead  from  anthrax. 

It  has  been  said  that  Hoffa's  work  was  the  first  suc- 
cessful attempt  to  study  the  chemical  poisons  of  anthrax. 
However,  his  results  cannot  be  considered  altogether  satis- 
factory.    The  small  amount  of  the  basic  substance  which 


ANTHRAX,  103 

he  obtained  rendered  it  highly  probable  that  in  the  case  of 
a  germ  so  virulent  as  that  of  anthrax  there  must  be  other 
chemical  poisons  produced.  This  supposition  has  been  con- 
firmed by  the  labors  of  Hankin,  who,  in  1889,  while  at 
work  in  Koch's  laboratory,  prepared  from  cultures  of 
the  bacillus  anthracis  an  albumose  which,  when  employed 
in  comparatively  large  amount,  proved  fatal  to  animals, 
but  when  used  in  very  small  quantity  gave  immunity 
against  subsequent  inoculations  with  the  living  germ. 
Unfortunately,  Han  kin  does  not  mention  the  symptoms 
induced  by  toxical  doses  of  this  substance.  Whether  or 
not  the  albumose  of  Hankin  contains  in  statu  nascendi 
the  base  of  Hoffa,  and  owes  its  poisonous  properties  to 
the  same,  has  not  been  determined. 

Brieger  and  Frankel  obtained  the  poisonous  proteid 
of  anthrax  from  animals  in  which  the  disease  had  been 
induced  by  inoculation  with  the  bacillus.  The  liver,  spleen, 
lungs,  and  kidneys  of  these  animals  were  finely  divided 
and  rubbed  up  with  water.  After  this  had  stood  in  a 
refrigerator  for  twelve  hours  it  was  passed  through  a 
Chamberland  filter  and  the  proteid  precipitated  from  the 
filtrate  with  absolute  alcohol. 

Martin,  by  growing  the  anthrax  bacillus  for  from  ten 
to  fifteen  days  in  an  alkaline  albuminate  from  blood  serum 
and  filtration  through  porcelain,  obtains  the  following 
metabolic  products  : 

1.  Protoalbumose  and  deuteroalbumose  and  a  trace  of 
peptone.  All  of  these  react  chemically  like  similar  sub- 
stances prepared  by  peptic  digestion. 

2.  An  alkaloid. 

3.  Small  quantities  of  leucin  and  tyrosin. 

The  most  characteristic  property  of  the  albumoses  is  that 
their  solutions  are  strongly  alkaline,  and  the  alkalinity  is 
not  removed  by  treatment  with  alcohol,  benzol,  chloroform, 
or  ether,  or  by  dialysis. 

The  alkaloid  is  soluble  in  water,  alcohol,  and  amylic 
alcohol ;  insoluble  in  chloroform,  ether,  and  benzol.  Its 
solutions  are  strongly  alkaline  and  the  alkaloid  forms  crys- 
talline salts  with  acids.     It  is  precipitated  by  the  general 


104  BACTEKIAL    POISONS. 

alkaloidal  reagents,  with  the  exception  of  potassio-mercuric 
iodide.  It  is  somewhat  volatile  and  loses  its  poisonous 
properties  on  exposure  to  the  air. 

The  mixed  albumoses  are  poisonous  only  in  considerable 
doses,  0.3  gramme  being  required  to  kill  a  mouse  of  22 
grammes  weight  when  injected  subcutaneously.  Smaller 
doses  cause  a  local  oedema  and  a  somnolent  condition,  from 
which  the  animal  recovers.  The  larger  doses  produce  a  more 
extensive  oedema  and  the  somnolence  deepens  into  coma, 
terminating  in  death.  In  some  cases  the  spleen  is  enlarged. 
The  absence  of  germs  was  demonstrated  by  plate  cultures. 
The  alkaloid  causes  similar  symptoms.  It  is,  however, 
more  poisonous  and  acts  more  rapidly  than  the  albumoses. 
The  animal  is  affected  immediately  after  the  injection,  and 
the  gradually  increasing  coma  terminates  in  death.  The 
alkaloid  also  produces  oedema,  and  in  many  cases  throm- 
bosis of  the  small  veins.  Extravasation  into  the  peritoneal 
cavity  is  occasionally  seen,  and  the  spleen  is  ordinarily 
enlarged  and  filled  with  blood.  The  fatal  dose  for  a  mouse 
is  from  0.1  to  0.15  gramme,  death  resulting  within  three 
hours. 

This  alkaloid  does  not  appear  to  be  identical  in  its  action 
with  the  anthracin  of  Hoffa. 

Asiatic  Cholera. — There  are  good  reasons,  apart  from 
experimental  evidence,  for  believing  that  the  comma  bacillus 
of  Koch  produces  its  ill  effects  by  the  elaboration  of  chemi- 
cal poisons.  This  germ  is  not  a  blood  parasite.  It  grows 
only  in  the  intestine,  and  the  symptoms  of  the  disease  and 
death  must  result  from  the  absorption  of  its  poisonous 
products.  In  confirmation  of  this  statement  experiment 
has  shown  that  this  is  one  of  the  most  active,  chemically, 
of  all. known  pathogenic  germs. 

In  the  first  place,  Bitter  has  shown  that  the  comma 
bacillus  produces  in  meat-peptone  cultures  a  peptonizing 
ferment,  which  remains  active  after  the  organism  has  been 
destroyed.  Like  similar  chemical  ferments,  it  converts  an 
indefinite  amount  of  coagulated  albumin  into  peptone.  It 
is    more  active  in  alkaline  than  in  acid  solutions,  thus 


ASIATIC    CHOLERA.  105 

resembling  pancreatin  more  than  pepsin.  This  resem- 
blance to  pancreatin  is  further  demonstrated  by  the  fact 
that  its  activity  is  increased  by  the  presence  of  certain 
chemicals,  such  as  sodium  carbonate  and  sodium  salicylate. 
That  a  diastatic  ferment  is  also  produced  by  the  growth  of 
the  bacillus  was  indicated  in  the  experiments  of  Bitter  by 
the  development  of  an  acid  in  nutrient  solutions  contain- 
ing starch  paste.  However,  all  attempts  to  isolate  the 
diastatic  ferment  were  unsuccessful.  A  temperature  of  60° 
destroys  or  greatly  decreases  the  activity  of  ptyalin,  and 
this  seems  to  be  true  also  of  the  diastatic  ferment  produced 
by  the  comma  bacillus.  But  the  formation  of  an  acid  from 
the  starch  pre-supposes  that  the  starch  is  first  converted  into 
a  soluble  form. 

Fermi  has  succeeded  in  isolating  the  peptonizing  ferment 
of  the  cholera  germ  in  the  following  manner :  65  per  cent, 
alcohol  added  to  gelatin  which  has  been  liquefied  by  the 
bacillus  precipitates  the  proteid,  but  not  the  ferment.  After 
twenty-four  hours  the  precipitate  is  removed  by  filtration 
and  the  ferment  precipitated  from  the  filtrate  by  the  addi- 
tion of  absolute  alcohol.  After  being  collected  on  a  filter 
and  dried  the  ferment  is  dissolved  in  an  aqueous  solution 
of  thymol  and  its  peptonizing  properties  demonstrated  on 
gelatin  tubes. 

Rietsch  believes  that  the  destructive  changes  observed 
in  the  intestines  in  cholera  are  due  to  the  action  of  the 
peptonizing  ferment. 

Cantani  injected  sterilized  cultures  of  the  comma  bacil- 
lus into  the  peritoneal  cavities  of  small  dogs  and  observed 
after  from  one-quarter  to  one-half  hour  the  following  symp- 
toms :  Great  weakness,  tremor  of  the  muscles,  drooping  of 
the  head,  prostration,  convulsive  contractions  of  the  pos- 
terior extremities,  repeated  vomiting,  and  cold  head  and  ex- 
tremities. After  two  hours  these  symptoms  began  to  abate, 
and  after  twenty- four  hours  recovery  seemed  complete. 
Control  experiments  with  the  same  amounts  of  uninfected 
beef-tea  were  made  with  negative  results.  The  cultures 
used  were  three  days  old  when  sterilized.  Older  cultures 
seemed  less  poisonous  and   a  high  or  prolonged  heat  in 


106  BACTERIAL    POISONS. 

sterilization  decreased  the  toxicity  of  the  fluid.  From  these 
facts  Cantani  concluded  that  the  poisonous  principle  is 
volatile,  but  the  effect  of  high  or  prolonged  heat  in  dimin- 
ishing the  toxicity  was  more  probably  due  to  its  destructive 
effect  on  the  poisonous  proteids. 

Cantani  also  observed  that  the  blood  of  those  sick  with 
cholera  is  acid  :  this  has  been  confirmed  by  Strauss  by 
the  examination  of  the  blood  directly  after  death ;  and 
Ahrend  found  lactic  acid  in  the  strongly  acid  urine  of  a 
cholera  patient. 

Nicati  and  Rietsch  produced  fatal  effects  in  dogs  by 
injecting  cultures,  from  which  all  germs  had  been  removed 
by  filtration,  into  the  bloodvessels.  Later,  the  same  inves- 
tigators obtained  from  old  bouillon  cultures  containing 
peptone  a  poisonous  base.  Ermengen  also  showed  that 
cultures  after  filtration  through  a  Chamberland  filter  are 
poisonous. 

Klebs  has  attemped  to  answer  experimentally  the  ques- 
tion, In  what  way  does  the  cholera  germ  prove  harmful  ? 
Cultures  of  the  bacillus  in  fish  preparations  were  acidified, 
filtered,  the  filtrate  evaporated  on  the  water-bath,  the  residue 
taken  up  with  alcohol  and  precipitated  with  platinum  chlo- 
ride. The  platinum  was  removed  with  hydrogen  sulphide, 
and  the  crystalline  residue  obtained  on  evaporation  was 
dissolved  in  Avater  and  injected  intravenously  into  rabbits. 
Muscular  contractions  were  induced.  Death  followed  in 
one  animal,  which,  in  addition  to  the  above  treatment, 
received  an  injection  of  a  non-sterilized  culture.  In  this 
case  there  was  observed  an  extensive  calcification  of  the 
epithelium  of  the  uriniferous  tubules.  Klebs  believes  this 
change  in  the  kidney  to  be  induced  by  the  chemical  poison, 
and  from  this  standpoint  he  explains  the  symptoms  of 
cholera  as  follows  :  The  cyanosis  is  a  consequence  of  arte- 
rial contraction,  the  first  effect  of  the  poison.  The  mus- 
cular contractions  also  result  from  the  action  of  the  poison. 
The  serous  exudate  into  the  intestines  follows  upon  epithe- 
lial necrosis.  Anuria  and  the  subsequent  symptoms  appear 
when  the  formation  and  absorption  of  the  poison  become 
greatest. 


ASIATIC    CHOLERA.  107 

Hueppe  states  that  the  severe  symptoms  of  cholera  can 
be  explained  only  on  the  supposition  that  the  bacilli  produce 
a  chemical  poison,  and  that  this  poison  resembles  muscarine 
in  its  action. 

Villiers  isolated  by  the  Stas-Otto  method  from  two 
bodies  dead  from  cholera,  a  poisonous  base  which  was  liquid, 
pungent  to  the  taste,  and  possessed  the  odor  of  hawthorn. 
It  was  strongly  alkaline,  and  gave  precipitates  with  the 
general  alkaloidal  reagents.  From  one  to  two  milligrammes 
of  this  substance,  injected  into  frogs,  caused  decreased 
activity  of  the  heart,  violent  trembling,  and  death.  The 
heart  was  found  in  diastole,  and  full  of  blood,  and  the 
brain  slightly  congested.  However,  the  presence  of  this 
substance  in  the  bodies  of  persons  who  have  died  of  cholera 
does  not  prove  that  its  production  is  due  to  the  cholera 
bacillus. 

Pouchet  extracted  from  cholera  stools,  with  chloroform, 
an  oily  base  belonging  to  the  pyridine  series.  It  readily 
reduces  ferric  as  well  as  gold  and  platinum  salts,  and  forms 
an  easily  decomposable  hydrochloride.  It  is  a  violent  poison, 
irritating  the  stomach,  and  retarding  the  action  of  the  heart. 
Subsequently,  he  obtained  an  apparently  identical  substance 
from  cultures  of  Koch's  comma  bacillus. 

In  1887,  Brieger  made  a  report  of  his  studies  on  the 
chemistry  of  the  cholera  bacillus.  He  used  pure  cultures  on 
beef-broth  (fleischbrei),  which  was  rendered  alkaline  by  the 
addition  of  a  3  per  cent,  soda  solution.  These  were  kept 
at  from  37°  to  38°.  After  twenty-four  hours,  cadaverine 
was  found  to  be  present.  Older  cultures  furnished  very 
small  quantities  of  putrescine,  but  cultures  on  blood-serum 
yielded  much  larger  amounts  of  this  base.  While  cada- 
verine and  putrescine  cannot  be  said  to  be  poisonous,  they 
do  cause  necrosis  of  tissue  into  which  they  are  injected,  and 
their  formation  by  the  cholera  bacillus  may  account  for  the 
necrotic  tissue  in  the  intestine  in  the  disease.  The  lecithin 
of  the  beef- broth  was  slowly  acted  upon  by  the  germs,  but 
with  age  the  amount  of  choline  increased,  reaching  its 
maximum  during  the  fourth  week. 

Creatine  proved  still  more  resistant  to  the  action  of  the 


108  BACTERIAL    POISONS. 

germs ;  but,  after  six  weeks,  a  considerable  quantity  of 
creatinine  was  isolated,  and  a  smaller  amount  of  methyl- 
guanidine.  The  latter  is  very  poisonous,  causing  muscular 
tremors  and. dyspnoea.  The  presence  of  methyl-guanidine 
indicates  that  the  comma  bacillus  acts  as  an  oxidizing  agent, 
since  creatine  yields  methyl-guanidine  only  by  oxidation. 

Brieger  succeeded  in  finding,  in  addition  to  the  above- 
mentioned  ptomaines,  which  are  common  products  of  putre- 
faction, two  poisons  which  he  considers  as  specific  products 
of  the  comma  bacillus.  One  of  these,  found  in  the  mer- 
curic chloride  precipitate,  is  a  diamine,  resembling  trime- 
thylenediamine.  It  produced  muscular  tremor  and  heavy 
cramps.  In  the  mercury  filtrate  was  found  another  poison, 
which,  in  mice,  produced  a  lethargic  condition  ;  the  respira- 
tion and  heart's  action  became  slow,  and  the  temperature 
sank,  so  that  the  animal  felt  cold.  Sometimes  there  was 
bloody  diarrhoea. 

Brieger  and  Frankel  found  that  the  insoluble  proteid 
which  they  obtained  from  cultures  of  the  cholera  bacillus, 
when  suspended  in  water  and  injected  subcutaneously  in 
guinea-pigs,  caused  death  after  from  two  to  three  days. 
Section  showed  inflammatory  swelling  and  redness  of  the 
subcutaneous  tissue,  extending  into  the  muscles  for  some 
distance  about  the  point  of  injection,  but  no  necrosis. 
There  was  no  change  in  the  intestines  and  no  effusion  into 
the  peritoneum.  In  some  instances  there  were  evidences 
of  beginning  fatty  degeneration  of  the  liver.  Upon  rab- 
bits this  substance,  even  in  large  doses,  was  without  effect. 

In  endeavoring  to  obtain  immunity  in  guinea-pigs  against 
cholera,  Gamaleia  employs  cultures  which  have  been  ster- 
ilized at  120°.  Subcutaneous  injections  of  these  cause 
transient  oedema,  and  the  animals  soon  recover.  The  high 
temperature  destroys  not  only  the  bacillus,  but  renders  inert 
certain  "  ferment-like  "  products.  However,  if  the  cultures 
be  sterilized  at  60°,  large  doses  (10  c.c.  per  kilogramme,  body 
weight)  cause  death,  injected  intravenously  in  rabbits,  and 
a  less  amount  produces  marked  symptoms.  The  animals 
refuse  food,  and  a  diarrhoea,  which  may  continue  for  hours, 
appears;    Often  there  is  cloudiness  of  the  cornea  and  reten- 


ASIATIC    CHOLERA.  109 

tion  of  urine,  which  is  albuminous.  The  animals  recover 
very  slowly.  In  this  connection  Bouchard  remarks  that 
in  1884  he  obtained  by  the  intravenous  injection  of  the 
urine  of  a  cholera  patient  in  rabbits  muscular  tremor,  cyan- 
osis, albuminuria,  and  diarrhoea,  but  that  he  has  never  suc- 
ceeded in  inducing  these  symptoms  with  the  cholera  vibrio. 

Petri  finds  that  the  comma  bacillus  produces  in  solu- 
tions of  peptone  large  amounts  of  tyrosin  and  leucin,  a 
small  quantity  of  indol,  fatty  acids,  poisonous  bases,  and  a 
poisonous  proteid.  The  proteid  resembles  peptone  in  its 
behavior  toward  heat  and  chemical  reagents,  and  is  desig- 
nated by  Petri  as  "  toxopeptone."  It  is  not  precipitated 
by  heat  or  concentrated  nitric  acid,  nor  by  potassium  ferro- 
cyanide  and  acetic  acid,  nor  by  ammonium  sulphate  added 
to  saturation.  With  sodium  phospho-tungstate  it  gives  a 
precipitate  which  clears  up  on  the  application  of  heat.  The 
precipitate  with  tannic  acid  is  insoluble  in  an  excess  of  the 
precipitant.  It  gives  the  biuret  reaction  perfectly,  but 
responds  to  Millon's  test  but  feebly. 

In  quantities  of  0.36  of  a  gramme  per  kilogramme  and 
more  it  is  fatal  to  guinea-pigs  within  eighteen  hours.  It 
produces  muscular  tremor  and  paralysis.  Post-mortem 
shows  an  effusion  into  the  peritoneal  cavity,  marked  injec- 
tion of  the  bloodvessels  of  the  intestines,  and  isolated 
hemorrhagic  spots. 

This  proteid  is  not  rendered  inert  by  a  temperature  of 
100°.  Petri  does  not  claim  that  he  has  obtained  a  chemi- 
cally pure  body,  but  supposes  that  it  is  contaminated  with 
more  or  less  unchanged  peptone. 

Scholl  has  studied  the  chemical  products  of  the  cholera 
bacillus  when  grown  under  anaerobic  conditions.  Fresh 
eggs  were  sterilized  and  inoculated  in  the  usual  way.  The 
eggs,  after  being  kept  for  eighteen  days  at  36°,  were  opened. 
The  contents  smelled  intensely  of  hydrogen  sulphide,  but 
not  of  amines.  The  albumin  was  completely  fluid,  while 
the  yolk  was  more  solid  and  of  a  dark  color. 

Five  c  c.  of  the  fluid  contents  were  injected  into  the 
abdomen  of  a  guinea-pig.  Soon  the  posterior  extremi- 
ties were  paralyzed,  and  after  ten  minutes  the  paralysis 


110  BACTERIAL    POISONS. 

became  general,  the  animal  lying  on  the  side.  After  five 
minutes  more  convulsive  movements  of  the  extremities 
began,  and  forty  minutes  after  the  injection  the  animal  was 
dead.  Section  showed  the  vessels  of  the  small  intestines 
and  stomach  highly  injected,  a  colorless  effusion  in  the 
peritoneal  cavity,  and  the  heart  in  diastole. 

The  albuminous  content  of  the  egg  was  poured  into  ten 
times  its  volume  of  absolute  alcohol.  The  precipitate  was 
collected  and  washed  with  alcohol  until  a  colorless  filtrate 
was  obtained.  The  precipitate  was  then  digested  for  fif- 
teen minutes  with  200  c.c.  of  water  and  filtered.  Eight 
c.c.  of  the  filtrate  was  injected  into  the  abdomen  of  a 
guinea-pig.  Paralysis  resulted  immediately,  and  within 
one  and  one-fourth  minutes  the  animal  was  dead.  Section 
showed  marked  injection  of  the  vessels  of  the  small  intes- 
tines, a  bloody  transudate  in  the  peritoneal  cavity  and  the 
heart  in  diastole. 

The  poisonous  proteid  was  rendered  inert  by  a  tempera- 
ture of  100°;  it  was  not  altered  by  short  exposure  to  75°, 
but  attempts  to  evaporate  the  solution  at  40°  in  vacuo  over 
calcium  chloride  destroyed  the  poisonous  properties.  The 
proteid  was  finally  precipitated  from  its  aqueous  solution 
by  a  mixture  of  alcohol  and  ether.  It  was  washed  with 
ether  and  the  ether  allowed  to  evaporate  spontaneously.  A 
small  bit  of  this  proteid  proved  fatal  to  guinea-pigs,  and 
the  same  post-mortem  changes  were  found  as  given  above. 
Scholl  classes  this  proteid  among  the  peptones.  It  is  not 
precipitated  by  heat  or  concentrated  nitric  acid,  singly  or 
combined,  nor  by  ammonium  sulphate.  It  gives  the  xantho- 
proteid  and  biuret  reactions.  Scholl  regards  this  as  the 
true  poison  of  cholera,  and  points  out  its  difference  from 
the  proteid  of  Brieger  and  Frank  el  and  that  of 
Petri. 

Bujwid  found  that  on  the  addition  of  from  five  to  ten 
per  cent,  of  hydrochloric  acid  to  bouillon  cultures  of  the 
cholera  bacillus  there  was  developed  after  a  few  minutes  a 
rose-violet  coloration  which  increased  during  the  next  half 
hour  and  in  a  bright  light  showed  a  brownish  shade.  The 
coloration  is  more  marked  if  the  culture  is  kept  at  about 


ASIATIC    CHOLERA.  Ill 

37°.  In  impure  cultures  this  reaction  does  not  occur. 
The  Finkler-Prior  bacillus  cultures  give  after  a  longer 
time  a  similar,  but  more  of  a  brownish  coloration.  Cul- 
tures of  many  other  bacilli  were  tried  and  failed  to  give 
this  reaction.1 

Brieger  found  that  this  color  is  due  to  an  indol  deriva- 
tive. In  cholera  cultures  on  albumins  he  obtained  indol 
by  distillation  with  acetic  acid. 

Bujwid  has  made  a  further  contribution  to  our  knowl- 
edge of  the  "  cholera-reaction."  His  conclusions  are  as 
follows  : 

(1)  Five  to  ten  per  cent,  of  hydrochloric  acid  added  to 
cholera  cultures  produce  a  rose-violet  coloration,  which  is 
characteristic  of  the  comma  bacillus. 

(2)  No  other  bacterium  gives  the  same  coloration  under 
the  same  conditions. 

(3)  The  coloration  appears  in  such  cultures  which  are 
from  ten  to  twelve  hours  old,  so  that  this  test  can  be  used 
for  diagnostic  purposes,  and  will  give  results  before  they 
can  be  obtained  by  plate  cultures. 

(4)  Impure  cultures  do  not  give  this  reaction. 

Dunham  finds  the  best  medium  for  the  "  cholera-reac- 
tion" to  be  a  one  per  cent,  alkaline  peptone  solution  with 
one-half  per  cent,  of  common  salt.  Bujwid  prefers  a  two 
per  cent,  feebly  alkaline  peptone  solution  with  salt.  Jadas- 
sohn finds  that  gelatin  cultures  give  the  reaction  both 
before  and  after  the  liquefaction  of  the  gelatin.  The  un- 
dissolved gelatin,  after  the  addition  of  hydrochloric  or 
sulphuric  acid,  becomes  rose-violet. 

Cohen  claims  that  cultures  of  other  bacilli  give  a  similar 
coloration,  but  Bujwid  explains  that  the  results  obtained 
by  Cohen  were  due  to  the  use  of  impure  acids,  which  con- 
tained nitrous  acid.  Salkowski  agrees  with  Bujwid,  and 
states  that,  when  acids  wholly  free  from  nitrous  acid  are 
used,  the  reaction  is  characteristic  of  the  comma  bacillus. 
He  explains  the  reaction  by  supposing  that  the  germ  pro- 

1  Poehl  deserves  the  credit  of  being  the  first  to  call  attention  to  this 
reaction,  though  his  work  was  evidently  unknown  to  Bujwid  at  the  time 
when  the  latter  published  his  report. 


112  BACTERIAL    POISONS. 

duces  nitrous  acid,  which  exists  in  the  culture  as  a  nitrite. 
On  the  addition  of  an  acid  the  nitrous  acid  is  set  free,  and 
acting  upon  the  indol,  which  is  also  present,  gives  the 
coloration. 

From  a  very  exhaustive  research  on  the  importance  of 
this  test  Petri  comes  to  the  following  conclusions  : 

(1)  Seven  pure  cultures  of  the  cholera  germ  from  as  many 
sources  gave  the  reaction  with  equal  distinctness. 

(2)  Of  one  hundred  other  bacteria  tested  in  the  same 
way  twenty  gave  a  red  coloration.  In  nineteen  of  these 
the  coloration  is  due  to  the  nitroso-indol  reaction  of 
Baeyer.  The  twentieth  (anthrax)  gave  a  color  which  is  not 
due  to  indol. 

(3)  In  case  of  the  cholera  germ  and  the  others  as  well, 
the  reaction  is  due  to  the  reducing  action  of  the  bacteria  on 
nitrates.  The  reaction  is  most  marked  at  blood-tempera- 
ture and  with  the  cholera  bacillus  ;  it  is  least  distinct  with 
the  bacilli  of  Finkler  and  Miller. 

(4)  None  of  these  bacteria  convert  ammonia  into  nitrite. 

(5)  The  simple  addition  of  sulphuric  acid  is  sufficient  to 
give  the  test,  which,  however,  is  most  marked  when  the 
nutritive  solution  contains  0.01  per  cent,  of  nitrate. 

(6)  The  reaction  is  most  marked  if  the  sulphuric  acid  be 
added  after  the  addition  of  a  very  dilute  nitrite  solution. 

Schuchardt  calls  attention  to  the  fact  that  Yirchow 
observed  a  red  coloration  on  the  addition  of  nitric  acid  to 
filtered  cholera  stools  in  1846.  Griesinger,  in  1885, 
also  made  mention  of  the  production  of  a  red  coloration  in 
rice-water  stools  on  the  addition  of  nitric  acid. 

A  "cholera-blue"  has  also  been  observed  by  Brieger 
in  cultures  in  meat  extract  containing  peptone  and  gelatin. 
This  substance,  which  is  yellow  by  reflected,  and  blue  by 
transmitted  light,  is  developed  by  the  addition  of  concen- 
trated sulphuric  acid  to  the  culture.  It  may  be  separated 
from  the  "cholera-red"  as  follows  :  Treat  the  culture  with 
sulphuric  acid,  then  render  alkaline  with  sodium  hydrate, 
and  extract  with  ether.  Evaporate  the  ether,  and  remove 
the  "  cholera-red "  with  benzol,  then  again  dissolve  the 
"  cholera-blue "  in   ether.     The   characteristic   absorption 


TETANUS.  113 

bands  for  this  coloring  matter  begin  in  the  first  third  of  the 
spectrum,  between  E  and  T1,  and  darken  all  of  the  zone 
lying  beyond. 

Winter  and  Lesage  treat  a  bouillon  culture  of  the 
cholera  germ  with  sulphuric  acid,  dissolve  the  precipitate 
in  an  alkaline  medium,  reprecipitate  with  acid,  and  redis- 
solve  in  ether,  which  on  evaporation  leaves  oily  drops, 
which,  on  cooling,  form  a  yellow  mass  of  the  appearance 
of  a  fat  This  substance  is  insoluble  in  water  and  acids, 
soluble  in  alkalies  and  ether.  It  melts  at  50°,  and  does 
not  lose  its  virulence  on  being  boiled  with  alcohol  rendered 
feebly  alkaline.  The  virulence  of  a  culture  and  the  amount 
of  this  substance  contained  therein  are  in  direct  proportion 
to  each  other. 

Small  doses  of  this  substance  (1  milligramme  to  100 
grammes  of  body  weight  of  the  animal)  in  feebly  alkaline 
solution  introduced  into  the  stomachs  of  guinea-pigs  cause, 
as  a  rule,  within  from  four  to  six  hours,  a  chill,  and  death 
after  twenty-four  hours.  With  larger  doses  the  tempera- 
ture falls  after  from  one-half  to  one  hour,  and  death  results 
within  from  twelve  to  twenty  hours.  Smaller  doses  cause 
a  less  marked  reaction  and  the  animal  recovers  within 
twenty-four  hours.  If  killed  within  this  time  the  animal 
shows  a  choleraic  condition.  Rabbits  succumb  only  after 
repeated  subcutaneous  injections.  The  substance  can  be 
extracted  from  the  muscles,  liver,  kidneys,  and  urine  of 
the  poisoned  animals.  It  can  also  be  obtained  from  cultures 
of  a  cholera  infantum  germ.  The  fact  that  this  poison  be- 
longs neither  to  the  ptomaines  nor  albumins  is  of  interest. 

Cunningham  describes  ten  species  of  the  common  ba- 
cillus, one  of  which  does  not  liquefy  gelatin,  and  fails  to 
respond  to  the  cholera  reaction.  He  also  states  that  there 
are  cases  of  undoubted  cholera  in  Calcutta  in  which  the 
common  bacillus  is  wholly  wanting. 

Tetanus. — In  1884,  Nicolaier,  by  inoculating  140 
animals  with  earth  taken  from  different  places,  produced 
symptoms  of  tetanus  in  69  of  them.  In  the  pus  which 
formed  at  the  point  of  inoculation  he  found  micrococci  and 


114  BACTERIAL    POISONS. 

bacilli.  Among  the  latter  was  one  which  was  somewhat 
longer  and  slightly  thicker  than  the  bacillus  of  mouse- 
septicsemia.  In  the  subcutaneous  cellular  tissue  he  found 
this  bacillus  alone,  but  could  not  detect  it  in  the  blood, 
muscles,  or  nerves.  Heating  the  soil  for  an  hour  rendered 
the  inoculations  with  it  harmless.  In  cultures,  Nicolaier 
was  unable  to  separate  this  bacillus  from  other  germs,  but 
inoculations  with  mixed  cultures  produced  tetanus.  In  the 
same  year,  Carle  and  Raton e  induced  tetanus  in  lower 
animals  by  inoculations  with  matter  taken  from  a  pustule 
on  a  man  just  dead  from  tetanus.  In  1886,  Rosenbach 
made  successful  inoculations  on  animals  with  matter  taken 
from  a  man  who  had  died  from  tetanus  consequent  upon 
gangrene  from  frozen  feet.  With  bits  of  skin  taken  from 
near  the  line  of  demarcation,  he  inoculated  two  guinea-pigs 
on  the  thigh  ;  tetanic  symptoms  set  in  within  twelve  hours, 
and  one  animal  died  within  eighteen,  and  the  other  within 
twenty-four  hours.  The  symptoms  corresponded  exactly 
with  those  observed  in  the  "  earth  tetanus  "  of  Nicolaier, 
and  the  same  bacillus  was  found.  With  mixed  cultures  of 
this,  Rosenbach  was  also  able  to  cause  death  by  tetanus 
in  animals.  Beumer  had  under  observation  a  man  who 
died  from  lockjaw  following  the  sticking  of  a  splinter  of 
wood  under  his  finger-nail.  Inoculations  of  mice  and 
rabbits  with  some  of  the  dirt  found  on  the  wood  led  to 
tetanus.  The  same  observer  saw  a  boy  die  from  this  dis- 
ease following  an  injury  to  the  foot  from  a  sharp  piece  of 
stone.  White  mice  inoculated  with  matter  from  the  wound, 
and  those  inoculated  with  dirt  taken  from  the  boy's  play- 
ground, died  of  tetanus.  The  bacillus  of  Nicolaier  was 
again  detected.  Giordano  reports  the  case  of  a  man  who 
fell  and  sustained  a  complicated  fracture  of  the  arm.  He 
remained  on  the  ground  for  some  hours,  and  when  assist- 
ance came  the  muscles  and  skin  were  found  torn  and  the 
wounds  filled  with  dirt.  On  the  fifth  day  he  showed  symp- 
toms of  tetanus,  from  which  he  died  on  the  eighth  day. 
Inoculations  and  examinations  for  the  bacillus  were  again 
successful.  Ferrari  also  made  successful  inoculat'ons 
with  the  blood   taken  during:  life   from   a  woman  with 


TETANUS.  115 

tetanus  after  an  ovariotomy.  Hocksinger  has  confirmed 
the  above-mentioned  observations  by  carefully  conducted 
experiments,  the  material  for  which  was  furnished  by  a  case 
of  tetanus  arising;;  from  a  very  slight  injury  to  the  hand, 
the  wound  being;  filled  with  dirt.  Shakespeare  has  suc- 
ceeded in  inducing  tetanus  in  rabbits  by  inoculating  them 
with  matter  taken  from  the  medulla  of  a  horse  and  of  a 
mule,  both  of  which  had  died  from  traumatic  tetanus. 
These  uniform  observations  leave  no  room  to  doubt  that 
tetanus  is  often,  at  least,  due  to  a  a;erm  which  exists  in 
many  places  in  the  soil,  and  that  the  disease  is  transmissible 
by  inoculation. 

Bonomb  observed  nine  cases  of  tetanus  among  seventy 
persons  injured  by  the  falling  of  a  church  from  the 
earthquake  at  Bajardo.  The  bacillus  of  Nicoeaier  was 
detected  in  the  wounds,  and  animals  inoculated  with  the 
lime-dust  of  the  fallen  building  died  of  tetanus.  Of  many 
persons  injured  by  the  falling  of  another  church  at  the 
same  time,  none  had  tetanus,  and  animals  inoculated  with 
the  lime  from  this  church  suffered  no  inconvenience. 

The  same  experimenter  found  the  bacillus  in  the  wound 
of  a  sheep  which  died  from  tetanus  after  castration. 

Beumer  found  the  tetanus  bacillus  in  the  sloughing; 
tissue  of  the  umbilical  cord  of  a  child  which  was  taken  ill 
on  the  sixth  day  after  birth,  and  died  four  days  later  from 
tetanus.  From  this  he  concludes  that  tetanus  neonatorum 
and  "  earth  tetanus  "  are  identical,  and  advises  that  the  cord 
should  be  dressed  antiseptically. 

Kitasato  has  succeeded  in  isolating  the  bacillus  of 
Nicolaier  by  growing  the  mixed  cultures,  from  the  pus  of  a 
wound  on  a  man  who  died  from  tetanus,  at  a  high  tempera- 
ture (80°),  and  subsequently  developing  the  germ  under 
hydrogen.  The  bacillus  grows  only  in  the  absence  of  air, 
and  not  in  carbonic  acid.  It  develops  on  agar,  blood-serum, 
and  gelatin,  the  last  of  which  it  gradually  liquefies  with  the 
formation  of  gas.  The  growth  is  more  vigorous  when  the 
nutritive  medium  contains  from  1.5  to  2  per  cent  of  grape- 
sugar. 

In  1888  Beleanti  and  Pescarolo  found  in  the  pus  of 


116  BACTEEIAL    POISON'S. 

a  wound,  which  was  followed  by  tetanus,  a  bacillus  which 
they  believed  to  differ  morphologically  from  that  of  Nico- 
laier  and  Rosenbach,  and  which  in  pure  cultures  induces 
tetanus  in  animals.  The  number  of  animals  experimented 
upon  Avas  great  and  included  mice,  guinea-pigs,  frogs, 
rabbits,  pigeons,  geese,  sparrows,  a  chicken,  and  a  dog. 
The  pigeons,  chicken,  geese,  and  frogs  proved  immune. 
After  subcutaneous  injections  a  bloody  oedema  appeared  at 
the  place  of  inoculation  and  pus  formed  in  small  quantity. 
Paralysis  first  appeared  and  was  followed  by  convulsions 
and  opisthotonos.  Later  studies  lead  Belfanti  and  Pes- 
carolo  to  conclude  that  their  bacillus  is  really  that  of 
Nicolaier,  but  differing  somewhat  from  that  of  Kita- 
SATO.  Kitasato  states  positively  that  the  germ  which  he 
has  isolated  is  absolutely  anaerobic,  while  the  Italians  find 
that  theirs  will  not  only  grow  aerobically,  but  when  so 
grown  will  induce  a  classical  tetanus. 

Lampiasi  found  in  the  blood  from  various  organs  of  a 
man  who  died  from  so-called  spontaneous  tetanus,  and  in 
two  cases  of  tetanus  in  mules,  a  spore-forming  bacillus, 
which  in  pure  cultures  induced  tetanus  in  animals.  This 
bacillus  is  wholly  different  morphologically  from  that  of 
Nicolaier. 

Widenmann  reports  a  very  interesting  case  of  a  boy 
who  fell  from  a  wall  and  wounded  his  face  on  a  piece  of 
vine-stake  in  the  earth.  The  boy  died  of  tetanus,  and  the 
splinters  extracted  from  the  face  and  the  earth  about  the 
stake  were  examined.  The  splinter  was  introduced  under 
the  skin  of  a  mouse,  which  died  thirty  hours  later  of  tetanus. 
In  the  pus  formed  about  the  splinter  numerous  microorgan- 
isms, among  which  a  micrococcus  and  a  short,  thick  bacillus 
abounded,  were  found,  but  in  none  of  the  many  animals  ex- 
perimented upon  could  the  bacillus  of  Nicolaier  be  de- 
tected. In  animals  inoculated  with  the  earth,  however,  the 
Nicolaier  germ  was  found.  Widenmann  concludes  that 
the  so-called  tetanus  bacillus  is  found  in  most  cases  on  ac- 
count of  its  very  wide  distribution  in  the  soil  and  not  as  a 
result  of  its  causal  relation  to  the  disease. 

Flugge  has  produced  tetanus  in  animals  without  being 


TETANUS.  117 

able  to  find  the  bacillus  of  Nicolaier,  and  Wyssokow- 
itsch  has  examined  an  earth  which  did  not  induce  tetanus, 
but  which  caused  suppuration,  and  in  the  pus  the  Nico- 
laier  bacillus  was  found  to  be  abundant.  With  the  pus 
obtained  from  three  cases  of  tetanus  neonatorum  due  to 
omphalitis  Kischensky  induced  tetanus  in  animals.  The 
pus  contained  pyogenetic  micrococci  and  a  short  bacillus, 
but  the  germ  of  Nicolaier  could  not  be  detected. 

Although  Kitt  claims  that  his  tetanus  bacillus  is  iden- 
tical with  that  of  Kitasato  (which  is  now  regarded  as  a 
pure  culture  of  the  germ  of  Nicolaier),  the  former  lique- 
fies solid  blood-serum  and  the  latter  does  not.  Bacteriolo- 
gists generally  agree  that  the  Nicolaier  bacillus  is  found 
only  at  the  place  of  inoculation  and  that  it  is  never  present 
in  the  blood  or  internal  organs,  yet  Shakespeare,  as  we 
have  seen,  induced  tetanus  in  rabbits  by  inoculating  them 
with  matter  taken  from  the  medulla  of  a  horse  and  that  of 
a  mule,  both  of  which  had  died  of  tetanus.  The  bacillus 
which  has  been  so  well  studied  by  Tizzoni  and  Cattani 
has  certain  constant  biological  differences  from  that  of 
Kitasato. 

Pla  has  studied  eight  cases  of  traumatic  tetanus  both  by 
cultures  and  by  inoculation  of  animals.  In  none  has  he 
found  the  germ  of  Nicolaier.  Moreover,  since  tetanus 
was  induced  in  animals  by  bits  of  matter  taken  from  the 
spinal  cord,  the  Nicolaier  germ  could  not  have  been  the 
cause,  if,  as  bacteriologists  now  teach,  this  germ  is  never 
found  save  at  the  place  of  inoculation. 

Brieger  has  obtained  in  the  mixed  cultures  of  the  germ 
of  Nicolai  er  and  Rosenbach  four  poisonous  substances. 
The  first,  tetanine,  which  rapidly  decomposes  in  acid  solu- 
tions, but  is  stable  in  alkaline  solutions,  produces  tetanus 
in  mice  when  injected  in  quantities  of  only  a  few  milli- 
grammes. The  second,  tetanotoxine,  produces  first  tremor, 
then  paralysis  followed  by  severe  convulsions.  The  third, 
to  which  no  name  has  been  given,  causes  tetanus  accom- 
panied by  free  flow  of  the  saliva  and  tears.  The  fourth, 
spasmotoxine,  induces  heavy  clonic  and  tonic  convulsions. 

Brieger  has  also  isolated  tetanine  from  the  amputated 


118  BACTERIAL    POISONS. 

arm  of  a  man  with  tetanus,  thus  showing  that  this  chemical 
poison  is  formed  in  the  body  as  well  as  in  the  artificial 
cultures. 

Brieger  and  Frankel  obtained  a  "  toxalbumin  "  from 
a  culture  of  Kitasato's  germ  in  bouillon  containing  grape- 
sugar.  This  substance  is  soluble  in  water,  and  when  in- 
jected in  small  amounts  subcutaneously  in  guinea-pigs, 
tetanus  appears  in  about  four  days,  and  soon  terminates 
fatally.  On  the  other  hand,  cultures  of  the  bacillus  of 
Tizzoni  and  Cattani  in  bouillon  with  sugar  fail  to  pro- 
duce any  chemical  poison,  but  the  cultures  in  gelatin  are 
highly  poisonous  after  filtration  through  porcelain.  Even 
one-half  cubic  centimetre  of  the  latter  induces  the  disease 
and  death  in  rabbits  weighing  from  one  and  a  half  to  two 
kilogrammes.  Death  results  never  later  than  three  days, 
while,  as  has  been  seen  above,  the  first  symptoms  induced 
by  the  poison  from  the  bacillus  of  Kitasato  usually 
appear  on  the  fourth  day.  Brieger  and  Frankel  ob- 
tained their  proteid  by  precipitation  with  absolute  alcohol, 
but  the  addition  of  this  agent  to  cultures  of  the  germ  of 
Tizzoni  and  Cattani  destroys  its  poisonous  properties. 
The  active  substance  of  the  Italian  germ  was  obtained 
either  (1)  by  dialysis,  solution  in  water,  and  evaporation  in 
a  vacuum ;  or  (2)  by  precipitation  with  ammonium  sul- 
phate, separation  by  dialysis,  and  drying  in  a  vacuum. 
This  poisonous  body  is  soluble  in  water,  non-dialyzable, 
destructible  by  a  temperature  above  60°,  and  by  treatment 
with  concentrated  mineral  acids,  and  is  unaffected  by  alka- 
lies or  by  prolonged  treatment  with  carbouic  acid  gas.  It 
contains  a  ferment  which  liquefies  gelatin  and  digests  fibrin. 
This  peptonizing  ferment  is  active  only  in  alkaline  solu- 
tion, and  is  present  in  the  bouillon  cultures  which  are  not 
poisonous ;  therefore,  the  poison  and  the  peptonizing  fer- 
ment must  be  two  distinct  bodies.  However,  on  account 
of  the  properties  which  we  have  mentioned,  Tizzoni  and 
Cattani  conclude  that  the  poison  also  belongs  to  the 
soluble  ferments  or  enzymes. 

Buschettini  has  studied  the  distribution  of  this  poison 


TETANUS.  119 

through  the  body  and  its  elimination  in    the    following 
manner : 

Animals  were  poisoned  by  injections  of  the  substance 
prepared  by  Tizzoni  and  Cattani,  and  just  before  death 
they  were  killed  and  bits  of  various  organs  rubbed  up  with 
sterilized  water  were  injected  into  other  animals.  Emul- 
sions from  the  liver  and  supra-renal  capsules  were  invariably 
without  effect,  while  those  from  the  kidney  were  constantly 
poisonous.  This  is  supposed  to  prove  that  the  poison  is 
eliminated  by  the  kidney.  The  blood  taken  from  the  vena 
cava  was  found  to  be  poisonous  in  three  out  of  four  experi- 
ments. When  the  injections  were  made  under  the  skin 
the  lumbar  cord  was  active  in  four  out  of  eight  cases,  and 
in  all,  when  the  injections  were  made  directly  into  the 
sciatic  nerve.  On  the  other  hand,  when  the  inoculations 
were  made  under  the  dura  mater,  the  brain  was  found  to 
be  active  while  the  lumbar  cord  remained  inactive.  From 
these  experiments  it  is  concluded  that  the  poison  uot  only 
circulates  in  the  blood,  but  is  deposited  in  the  central 
nervous  system. 

A.  Babes  prepared,  from  cultures  made  by  V.  Babes 
and  Puscaria  in  agar  containing  no  peptone,  an  albumose 
which  causes  tetanus  in  animals. 

Faber  finds  in  a  mixed  culture  a  poisonous  proteid 
body  which  resembles  closely,  so  far  as  it  has  been  studied, 
that  of  Tizzoni  and  Cattani.  Faber  lays  much  stress 
upon  the  arguments  in  favor  of  this  substance  being  a 
soluble  ferment.  With  this  proteid,  convulsive  movements 
first  appear  and  become  very  distinct  in  the  muscles  about 
the  point  of  injection.  In  case  very  small  amounts  are 
employed,  the  convulsive  movements  do  not  become  general 
and  the  animal  finally  recovers. 

Peyraud  claims  to  have  secured  immunity  in  animals 
against  "earth  tetanus"  by  giving  to  them  strychnia  in 
gradually  increased  doses.  Nocard  could  not  confirm  this 
claim. 

According  to  Led  antes,  the  poisonous  arrows  of  the 
natives  of  the  New  Hebrides  are  prepared  as  follows  :  The 
points,  which  are  usually  made  from  human  bones,  are  first 


120  BACTERIAL    POISONS. 

covered  with  a  vegetable  resin,  then  smeared  with  the  slime 
of  swampy  places. 

Liermann  found  that  material  taken  from  the  arm  of  a 
man  who  had  died  from  tetanus,  and  who  had  been  buried 
for  two  and  one-half  years,  induced  tetanus  in  animals. 
This  would  seem  to  show  that  the  poison  retains  its  viru- 
lence for  a  long  time.  In  this  material  there  were  found 
nine  kinds  of  bacteria,  but  none  of  these  in  pure  culture, 
or  in  mixed  culture,  induced  the  disease.  This  is  explained 
by  the  supposition  that  non-pathogenic  bacteria  may  receive 
toxicogenic  properties  from  the  media  in  which  they  grow. 

Tuberculosis. — Whatever  may  be  the  ultimate  verdict 
concerning  the  curative  properties  of  Koch's  tuberculin,  its 
employment  has  made  us  familiar  with  the  action  of  the 
chemical  products  of  the  bacillus  tuberculosis  on  man.  Un- 
fortunately, Koch  has  given  us  but  little  information  con- 
cerning the  nature  of  his  tuberculin,  and  the  little  which  he 
has  given  us  has  been  to  some  extent  misleading.  We 
would  not  imply  that  he  has  intentionally  been  misleading. 
Indeed,  we  believe  that  such  was  not  his  intention.  He 
speaks  of  the  agent  as  an  extract  of  a  pure  culture  of  the 
bacillus  tuberculosis  with  50  per  cent,  glycerin.  One  would 
infer  from  Koch's  statements  that  tuberculin  is  prepared 
by  extracting  the  bacterial  cells  with  50  per  cent,  glycerin, 
and  that  the  bacterial  products  are  not  present.  But, 
as  has  been  shown  by  Hueppe  and  Scholl,  the  proteids 
of  the  cells  of  the  bacillus  tuberculosis  cannot  be  extracted 
with  50  per  cent,  glycerin.  Moreover,  the  same  investiga- 
tors have  prepared  a  fluid  identical  in  physical  properties, 
in  chemical  reactions,  and  in  its  effects  on  animals,  with 
Koch's  fluid,  by  each  of  the  three  following  methods  : 

1.  Cultures  of  the  bacillus  are  filtered,  sterilized Jby  heat, 
and  concentrated. 

2.  The  supernatant,  fluid  portion  of  the  culture  is  de- 
canted from  the  mass  of  germs  at  the  bottom  of  the  flask, 
and  then  concentrated. 

3.  The  culture  is  freed  from  germs  by  filtration  through 
a  Chamberland  filter,  and  concentrated. 


TUBERCULOSIS.  121 

These  fluids  contain  :  1,  the  constituents  of  the  nutritive 
medium  which  have  not  been  altered  by  the  growth  of  the 
germ,  such  as  glycerin,  albumins,  albumoses,  and  peptones  ; 
2,  the  bacterial  products,  which  may  possibly  belong  to  the 
ptomaines,  the  bacterial  albumins  or  albumoses  and  bacterial 
ferments  ;  and  3,  any  constituents  of  dead,  broken-down 
bacilli  which  may  have  passed  into  solution.  To  which  of 
these  constituents  the  action  of  the  fluid  is  due  has  not  been 
positively  determined.  However,  from  the  similarity  in 
the  action  of  this  fluid  with  that  of  the  bacterial  products 
of  other  germs,  we  seem  justified  in  assuming  that  these 
constitute  the  active  principle. 

As  early  as  1888,  Hammerschlag  found  a  poisonous 
proteid  among  the  products  of  the  growth  of  this  germ. 
More  recently  he  finds  that  as  much  as  27  per  cent,  of  the 
cellular  substance  of  the  bacillus  tuberculosis  is  soluble  in 
alcohol  and  ether.  In  this  extract  there  is,  in  addition  to 
fat  and  lecithin,  a  poison  which  induces  in  rabbits  and 
guinea-pigs  convulsions  followed  by  death.  The  part  insol- 
uble in  alcohol  and  ether  consists  of  cellulose  and  proteids. 
Hammerschlag  has  also  prepared  from  cultures  of  this 
bacillus  a  "  toxalbumin "  which,  when  injected  subcuta- 
neously  in  rabbits,  causes  an  elevation  of  temperature  of 
from  1°  to  2°,  which  continues  for  a  day  or  longer. 

Zuelzer  has  reported  the  isolation  of  a  poisonous  pto- 
maine from  agar  cultures  of  the  bacillus  tuberculosis.  He 
says  that  the  injection  of  1  centigramme  or  less  of  this 
substance  subcutaneously  in  rabbits  or  guinea-pigs  causes, 
after  from  three  to  five  minutes,  increased  frequency  of 
respiration  (to  180  per  minute?)  and  an  elevation  of  tem- 
perature of  from  0.5°  to  1°.  He  also  reports  marked  pro- 
trusio  bulbi  as  a  constant  symptom  ;  the  eyes  become  very 
bright  and  the  pupils  are  dilated.  From  two  to  three 
centigrammes  suffice  to  kill  rabbits,  death  occurring  in  from 
two  to  four  days.  The  place  of  injection  is  reddened,  and 
hemorrhagic  spots  are  formed  in  the  mucous  membrane  of 
the  stomach  and  small  intestines.  In  two  instances  from 
15  to  20  cubic"  centimetres  of  clear  fluid  were  found  in  the 
peritoneal  cavity. 


122  BACTERIAL    POISON'S. 

Baumgarten  draws  the  following  conclusions  from  his 
experiments  with  tuberculin  on  rabbits  with  inoculation 
tuberculosis : 

It  causes  an  exudative  inflammation  in  the  vascular 
tissue  about  the  tubercle,  and  in  this  way  the  tuberculous 
tissue  may  be  isolated  and,  when  situated  superficially,  re- 
moved. In  some  cases,  however,  after  the  prolonged  employ- 
ment of  the  agent,  the  tuberculous  tissue  itself  may,  under 
the  influence  of  the  exudative  fluid  and  the  polynuclear 
leucocytes,  break  down  and  form  abscesses.  The  bacilli 
themselves  are  in  no  way  harmed  by  the  use  of  tuber- 
culin, and,  after  its  constant  employment  for  months,  they 
retain  their  original  form  and  lose  none  of  their  virulence. 
Some  preparations  seem  to  show  that  the  bacilli  multiply 
more  rapidly  when  the  injections  are  made,  but  a  positive 
statement  on  this  point  is  reserved  until  further  studies 
have  been  made.  It  is  certain,  however,  that  the  non- 
tubercular  tissue  of  animals  acquires  no  immunity  against 
the  disease  from  the  injections.  This  is  shown  by  the 
appearance  of  metastatic  foci  in  animals  in  which  from 
seven  to  twelve  grammes  of  the  original  lymph  (an  amount 
which  would  be  equivalent  to  from  seventy  to  one  hundred 
and  eighty  grammes  in  man)  has  been  injected.  It  is  further 
shown  by  the  fact  that  in  some  animals  treated  subcutane- 
ously,  tubercles  have  appeared  at  the  point  of  injection. 

Prudden  and  Hodenpyl  summarize  the  results  which 
they  have  obtained  by  the  inoculation  of  animals  with  dead 
tubercle  bacilli  as  follows :  "  These  dead  tubercle  bacilli 
are  markedly  chemotactic.  When  introduced  in  consider- 
able amount  into  the  subcutaneous  tissue  or  into  the  pleural 
or  abdominal  cavities,  they  are  distinctly  pyogenetic,  caus- 
ing aseptic  localized  suppuration.  Under  these  conditions 
they  are  capable,  moreover,  of  stimulating  the  tissues  about 
the  suppurative  foci  to  the  development  of  a  new  tissue, 
closely  resembling  the  diffuse  tubercle  tissue  induced  by 
the  living  germ.  We  have  found  that  dead  tubercle  bacilli 
introduced  in  small  numbers  into  the  bloodvessels  of  the 
rabbit  largely  disappear  within  a  few  hours  or  days,  but 
that  scattering  individuals  and  clusters  may  remain  here 


TUBERCULOSIS.  123 

and  there  in  the  lungs  and  liver,  clinging  to  the  vessel  walls 
for  many  days  without  inducing  any  marked  changes  in  the 
latter.  After  a  time,  however — earliest  in  the  lung,  later, 
as  a  rule,  in  the  liver — a  cell  proliferation  occurs  in  the 
vicinity  of  these  dead  germs,  which  leads  to  the  formation 
of  new  multiple  nodular  structures  bearing  a  striking  mor- 
phological resemblance  to  miliary  tubercles.  There  is  in 
them,  however,  no  tendency  to  cheesy  degeneration  and  no 
evidence  of  proliferation  of  the  bacilli,  but  rather  a  steady 
diminution  in  their  number.  It  seems  to  us  that  the  new 
structures  originate  in  a  proliferation  of  the  vascular  endo- 
thelium under  the  stimulus  of  the  dead  and  disintegrating 
germs." 

Maffucci  finds  that  cultures  of  the  tubercle  bacillus 
(from  a  mammal),  when  grown  from  one  to  six  months  on 
glycerin,  blood-serum,  or  liquid  blood-serum,  and  then 
sterilized  by  being  repeatedly  heated  to  from  65°  to  70°, 
produces  in  guinea-pigs,  when  employed  subcutaneously,  a 
progressive  marasmus,  which  terminates  fatally  within  from 
fourteen  days  to  five  or  six  months.  He  also  finds  that 
eggs  inoculated  with  sterilized  cultures  of  the  chicken  tuber- 
culosis bacillus  produce  chickens  which  are  feeble  and  soon 
die  of  emaciation.  In  neither  the  guinea-pigs  nor  chickens 
could  he  find  any  tubercles.  This  author,  unfortunately, 
does  not  state  positively  whether  the  bacilli  employed  in  his 
experiments  on  guinea-pigs  were  obtained  from  man  or  some 
other  mammal. 

Crookshank  and  Herroun  report  the  isolation  of  a 
ptomaine  and  an  albumose  not  only  from  artificial  cultures 
of  the  bacillus,  but  also  from  bovine  tuberculous  tissue. 
The  ptomaine  is  reported  as  causing  an  elevation  of  tem- 
perature in  tuberculous,  and  a  depression  in  healthy,  ani- 
mals. "  The  albumose,  whether  obtained  from  pure  culti- 
vations of  the  bacillus,  or  from  tuberculous  tissue,  produced 
a  marked  rise  of  temperature  in  tuberculous  guinea-pigs. 
On  the  other  hand,  in  an  experiment  tried  on  a  healthy 
guinea-pig,  there  was  an  equally  well-marked  fall  of  tem- 
perature." 


124  BACTERIAL    POISONS. 

Diphtheria. — That  the  Loffler  bacillus  is  a  cause 
of  diphtheria  no  one  can  now  deny.  The  fact  that  this 
germ,  although  found  only  at  the  seat  of  inoculation,  causes 
marked  systemic  disturbances,  indicates  that  its  action  must 
be  due  to  its  soluble  products.  This  was  early  recognized 
by  Loffler,  who  in  1887  attempted  to  ascertain  the 
nature  of  the  poison.  A  flask  of  bouillon  containing  pep- 
tone and  grape-sugar  was,  three  days  after  it  had  been 
inoculated  with  the  bacillus,  evaporated  to  10  c.c,  and  this 
was  injected  into  an  animal,  but  was  without  effect.  A 
second  flask  of  the  same  material  was  extracted  with  ether, 
but  this  extract  was  also  found  to  be  inert.  Next,  some 
neutral  beef  broth  was  extracted  with  glycerin  some  four 
or  five  days  after  it  had  been  inoculated  with  the  bacillus. 
The  glycerin  extract,  when  treated  with  five  times  its 
volume  of  absolute  alcohol,  deposited  a  voluminous,  floc- 
culent  precipitate,  which  was  collected,  washed  with  alcohol, 
dried,  and  dissolved  in  a  little  water.  A  further  precipita- 
tion with  alcohol  and  a  current  of  carbonic  acid  gas  secured 
a  white  substance,  and  the  injection  of  from  0.1  to  0.2 
gramme  of  this,  dissolved  in  water,  subcutaneously  in 
guinea-pigs,  caused  marked  pain  followed  by  a  fibrous 
swelling  with  hemorrhage  into  the  muscles  and  oedema, 
terminating  in  necrosis.  From  these  studies  Loffler 
concluded  that  the  poison  belongs  to  the  enzymes. 

Roux  and  Yersijst  found  that  bouillon  cultures  from 
which  the  bacillus  had  been  removed  by  filtration  through 
a  Chamberland  filter  are  poisonous,  especially  cultures 
which  are  four  or  five  weeks  old.  The  results  obtained 
varied  with  the  amount  of  the  fluid,  the  species  of  animal, 
and  the  method  of  administration.  The  effects  observed 
were  a  serous  exudation  into  the  pleural  cavity,  a  marked, 
acute  inflammation  of  the  kidney,  fatty  degeneration  of  the 
liver,  especially  after  injection  into  a  bloodvessel,  and  ©ede- 
matous swelling  in  the  surrounding  tissue  after  subcu- 
taneous inoculation.  In  some  instances,  in  dogs,  rabbits, 
and  guinea-pigs,  paralysis,  generally  in  the  posterior  extre- 
mities, followed.  The  action  of  the  poison  was  found  to 
be  very  slow,  and,  as  a  rule,  death  occurred  days,  and  in 


DIPHTHERIA.  125 

some  instances  weeks,  after  the  inoculation,  and  was  pre- 
ceded by  marked  emaciation . 

The  cultures  first  employed  were  seven  days  old ;  older 
cultures  (six  weeks)  contain  more  of  the  poison,  and  the 
symptoms  appear  within  a  few  hours.  In  cultures  espe- 
cially rich  in  the  poison,  a  small  amount  (from  0.2  to  2  c.c.) 
injected  under  the  skin  in  guinea-pigs  suffices  to  induce  the 
symptoms.  Mice  and  rats  are  markedly  insusceptible,  but 
succumb  to  large  doses. 

Heating  to  100°  for  twenty  minutes  renders  the  poison 
inert,  and  a  temperature  of  58°  maintained  for  two  hours 
markedly  lessens  its  virulence. 

The  poisonous  substance  is  precipitated  by  absolute 
alcohol,  and  is  carried  down  mechanically  on  the  addi- 
tion of  calcium  chloride  to  the  filtered  cultures.  These 
investigators  agree  with  Loffler  that  the  poison  belongs 
to  the  enzymes.  The  great  toxicity  of  this  substance  is 
indicated  by  the  statement  of  Roux  and  Yersin  that  0.4 
milligramme  suffices  to  kill  eight  guinea-pigs  or  two  rab- 
bits, and  that  2  centigrammes  of  the  calcium  chloride 
precipitate,  containing  about  0.2  milligramme  of  the  pure 
poison,  will  kill  a  guinea-pig  within  four  days. 

Brieger  and  Frankel  have  made  a  very  complete 
study  of  the  chemical  products  of  the  Loffler  bacillus. 
They  employed  cultures  of  bouillon  and  peptone  containing 
from  five  to  six  per  cent,  of  glycerin,  and  others  containing 
ten  per  cent,  of  sterile,  fluid  blood-serum.  The  latter  were 
found  to  be  most  suitable.  In  these  the  bacilli  grow  most 
abundautly.  In  all  cases  they  confirmed  the  statement  of 
Roux  and  Yersin  that  the  cultures,  at  first  alkaline,  be- 
come strongly  acid,  and  finally  again  alkaline,  with  the 
exception  that  the  glycerin  cultures  remained  acid. 

For  the  removal  of  the  bacteria  two  methods  were  em- 
ployed. First  the  bacilli  were  destroyed  by  heat.  When 
a  temperature  of  100°  was  employed  the  cultures  were 
rendered  inert,  but  it  was  found  that  exposure  for  from 
three  to  four  hours  to  a  temperature  of  50°  was  sufficient 
to  destroy  the  germs,  while  the  virulence  of  the  chemical 
products  was  not  affected.     The  second  method  of  removing 


126  BACTERIAL    POISONS. 

the  bacteria  consisted  of  filtration  through  a  Chamberland 
filter.  The  germ-free  filtrate  could  be  heated  to  50°  with- 
out loss  of  toxicity,  while  a  temperature  of  60°  rendered 
it  inert.  In  the  majority  of  the  experiments  the  filtration 
method  was  used  and  in  this  way  a  large  quantity  of  a 
poisonous  fluid  of  uniform  strength  was  obtained. 

Varying  amounts  of  this  fluid  were  used  upon  animals, 
mostly  guinea-pigs  and  rabbits,  and  it  was  found  that  the 
effects  varied  with  the  quantities  employed  and  the  methods 
of  administration.  The  symptoms  appeared  most  promptly 
when  the  injections  were  made  directly  into  a  bloodvessel. 
Of  four  rabbits  which  were  given  subcutaneously  respec- 
tively 1,  2|,  5,  and  10  c.c.  of  the  filtrate  on  December  the 
28th,  the  first  died  January  4th  ;  the  second,  January  2d  ; 
the  third,  December  31st ;  and  the  fourth,  December  30th. 
In  all  cases  in  which  death  did  not  occur  too  early,  paralysis 
appeared.  The  limbs  were  first  paralyzed,  and  this  was 
true  whether  the  fluid  was  administered  intravenously  or 
subcutaneously.  The  post-mortem  appearances  were  iden- 
tical with  those  observed  after  inoculation  with  the  bacillus, 
with  the  exception  of  the  absence  of  the  pseudo-membrane. 
After  subcutaneous  infection  there  was  a  gelatinous,  grayish- 
white,  sometimes  reddish,  cedematous  fluid  formed  at  the 
point  of  injection ;  and,  after  larger  doses,  necrosis.  In 
cases  in  which  death  was  delayed,  there  were  effusions  in 
the  pleura,  fatty  degeneration  of  the  liver,  and  inflamma- 
tion of  the  kidneys.  Especially  marked  were  these  cellular 
changes  in  rabbits  which  were  treated  with  small  amounts 
intravenously. 

Brieger  and  Frankel  conclude  this  part  of  their 
report  with  the  following  statement :  "  We  have  shown 
that  the  Loffler  diphtheria  bacillus  produces  in  its  cul- 
tures a  poisonous,  soluble  substance,  separable  from  the 
bacteria,  which  causes  in  susceptible  animals  the  same 
phenomena  which  are  induced  by  inoculation  with  the 
living  microorganism.  We  have  further  shown  that  this 
substance  is  destroyed  by  a  temperature  over  60°,  but  that 
it  can  be  heated  to  50°,  even  in  the  presence  of  an  excess 
of  hydrochloric  acid,  without  being  destroyed.     This  last 


DIPHTHERIA.  127 

fact  is  contrary  to  the  assumption  that  the  chemical  poison 
of  the  diphtheria  bacillus  is  a  ferment  or  enzyme." 

The  fluid  was  tested  for  basic  products,  but  with  wholly 
negative  results,  except  that  small  amounts  of  kreatinin  and 
cholin  were  found.  It  was  also  distilled  at  from  20°  to 
35°  in  a  vacuum,  and  the  distillate  was  found  to  be  inert. 

The  poisonous  substance  was  found  to  be  insoluble  in 
alcohol,  soluble  in  water,  and  non-dialyzable.  It  was  pre- 
cipitated by  saturation  with  ammonium  sulphate. 

The  substance  was  obtained  by  allowing  the  germ-free 
filtrate,  after  being  rendered  feebly  acid  with  acetic  acid,  to 
fall  into  a  large  volume  of  absolute  alcohol.  It  was  puri- 
fied by  repeated  solution  in  water  and  precipitation  with 
alcohol.  It  contains  a  large  amount  of  sulphur,  and  re- 
sponds to  the  biuret  and  Millon  tests.  It  is,  therefore, 
classified  among  the  albumins.  Since  it  is  not  precipitated 
by  saturation  with  magnesium  sulphate  at  30°,  it  cannot 
belong  to  the  globulins.  The  fact  that  it  is  precipitated  by 
saturation  with  ammonium  sulphate,  and  that  it  does  not 
dialyze,  shows  that  it  is  not  peptone.  It  is,  therefore, 
classified  by  Brieger  and  Frankel  among  the  albumins, 
and  is  designated  as  a  "  toxalbumin." 

The  special  reactions  and  the  results  of  an  ultimate 
analysis  of  this  substance  have  already  been  given  (page  20). 

This  proteid  induces  in  animals  all  the  symptoms  and 
post-mortem  appearances  which  have  been  mentioned  as 
following  the  administration  of  the  filtered  cultures.  It  is 
to  be  noted  that  the  injection  of  small  quantities  of  this 
proteid  (2J  milligrammes  per  1  kilogramme  of  the  body- 
weight  of  the  animal)  does  not  produce  its  effects  until  after 
the  lapse  of  weeks,  and  possibly  months.  This  peculiarity 
in  action  distinguishes  this  class  of  substances  from  all  other 
chemical  poisons,  and  it  has  received  as  yet  no  satisfactory 
explanation.  There  is  no  reason  for  believing  that  the  body 
obtained  by  Brieger  and  Frankel  is  chemically  pure, 
and  until  it  has  been  obtained  in  this  condition  we  can 
only  speculate  concerning  its  true  nature. 

It  should  be  remarked  that  the  Loffler  bacillus  shows 
not  only  marked  morphological  variations,  but  that   it  is 


128  BACTEKIAL    POISONS. 

very  variable  in  its  virulence,  some  cultures  having  been 
obtained  which  are  wholly  without  effect  upon  animals. 
From  cultures  of  this  kind  Brieger  and  Frankel  pre- 
pared a  non-poisonous  albumin  differing  in  its  ultimate 
composition  and  in  many  of  its  chemical  reactions  from  the 
poisonous  one. 

Frankel  has  been  unable  to  secure  immunity  in  ani- 
mals against  diphtheria  by  the  employment  of  small  doses 
of  the  "  toxalbumin."  If  the  dose  is  large  enough  the 
animal  dies.  If  it  is  smaller,  the  animal  seems  to  become 
more  susceptible  and  succumbs  more  readily  to  inoculations 
with  the  germ.  While  this  is  true  of  the  filtered  culture,  it 
is  not  the  case  with  that  which  has  been  sterilized  by  heat. 
Frankel  finds  that  if  from  10  to  20  c.c.  of  a  cul- 
ture of  the  bacillus  three  weeks  old,  which  has  been 
heated  for  one  hour  at  from  65°  to  70°,  be  injected  under 
the  skin  of  the  abdomen  of  guinea-pigs,  immunity  against 
subsequent  inoculation  with  the  virulent  germ  is  secured, 
provided  that  the  inoculation  is  not  made  earlier  than  the 
fourteenth  day  after  the  treatment  with  the  sterilized 
culture.  He  thinks  that  the  culture  contains  two  specific 
albumins,  one  of  which  is  poisonous,  while  the  other  gives 
immunity.  The  former  is  destroyed  by  a  temperature  of 
from  65°  to  70°,  while  the  other  retains  its  characteristic 
properties.  He  admits  the  possibility  that  the  poisonous 
albumin  may  be  converted  into  the  other  form  by  the  high 
temperature.  He  finds  that  the  modified  culture,  which 
gives  immunity,  is  of  no  service  for  therapeutic  purposes, 
and  that  if  an  animal  be  treated  with  it  directly  after  inocu- 
lation with  the  germ,  death  is  not  retarded,  but  is  hastened. 
From  these  experiments  he  concludes  that  the  vaccination 
albumin  at  first  lessens,  and  subsequently  increases  the 
resistance  of  the  animal. 

Sprouck  and  his  students  have  confirmed  the  above 
statements  concerning  the  toxicity  of  the  germ-free  cultures 
of  this  bacillus.  They  have  also  called  attention  to  the 
albuminuria  following  the  employment  of  this  poison.  In 
the  urine  they  find  casts,  white,  and  sometimes  red,  blood- 
corpuscles.     Microscopic  examination  of  the  kidney  after 


SUPPURATION.  129 

death  shows  the  same  changes  which  are  observed  in  the 
diphtheritic  nephritis  of  children.  Babes  also  finds  that 
the  germ-free  cultures  produce  the  parenchymatous  degener- 
ations of  the  internal  organs  which  are  found  in  the  human 
body. 

Tangl  has  shown  that  the  chemical  poison  is  formed  in 
the  body  as  well  as  in  culture-flasks.  A  large  piece  of 
pseudo-membrane  was  macerated  in  water  in  an  ice-chest 
for  twenty-four  hours,  and  then  filtered  through  porcelain. 
The  filtrate,  injected  into  animals,  produced  all  the  symp- 
toms which  have  been  obtained  by  a  similar  employment  of 
artificial  cultures.  Tangl  also  observed  that  in  some  cases 
in  which  the  animals  were  inoculated  with  the  sterilized 
culture  through  the  mucous  membrane  a  pseudo-membrane 
formed  at  the  point  of  injection. 

Suppuration. — As  early  as  1879,  Leber  concluded 
from  his  observation  on  infective  keratitis  that  the  asper- 
gillus  must  produce  certain  soluble  products  which  diffuse 
through,  the  cornea  and  set  up  an  inflammatory  action  in 
the  adjacent  vascular  tissue.  In  1882,  he  showed  that  sup- 
puration could  be  induced  by  the  introduction  of  sterilized 
mercury  and  copper,  and  that  the  pus  formed  is  free  from 
germs.  In  1884,  he  induced  suppuration  by  the  injection 
of  cultures  of  the  staphylococcus  pyogenes  aureus  which 
had  been  sterilized  by  being  boiled  for  hours.  In  1888, 
the  same  investigator  reported  that  he  had  found  an  alco- 
holic extract  of  the  dried  staphylococcus  to  be  highly  pyo- 
genetic.  From  this  extract  he  has  prepared  a  crystalline 
body  which  he  calls  phlogosin.  This  substance  is  readily 
soluble  in  alcohol  and  ether,  sparingly  soluble  in  water, 
and  it  crystallizes  in  needles.  The  crystals  can  be  sub- 
limed, leaving  no  residue,  and  the  sublimate,  which  forms 
in  rosettes,  still  possesses  the  pyogenetic  properties.  Alkalies 
precipitate  this  substance  from  its  solution  in  amorphous 
granules,  which. dissolve  in  acids,  forming  crystalline  salts. 
Leber  refers  to  the  observation  of  the  botanist  Pfeffer, 
who  found  that  vegetable  cells  are  attracted  by  certain 
chemical  substances,  and  adopts  the  term  chemotactic  action 


130  BACTERIAL    POISONS. 

(ehemotactisehe  Wirkung)  to  indicate  the  property  of  certain 
chemical  agents  of  attracting  leucocytes. 

As  has  been  stated,  Buchner  has  found  that  the  cells  of 
many  bacteria  contain  pyogenetic  proteids.  The  amount 
of  these  substances  in  the  cells  varies  with  the  kind  of 
germ,  and  some  species  (the  bacillus  prodigiosus,  for  in- 
stance) seem  to  contain  no  such  bodies.  The  bacillus  pyo- 
cyaneus  contains  a  large  quantity  of  the  proteid,  and  is 
suitable  for  lecture  demonstration.  The  germs  are  taken 
from  potato  cultures  and  rubbed  up  with  water.  Then 
they  are  treated  with  about  fifty  volumes  of  a  0.5  per  cent, 
solution  of  caustic  potash.  This  forms  in  the  cold  a  muci- 
laginous mass  which  dissolves  at  the  temperature  of  the 
water-bath.  After  being  heated  for  some  hours  the  fluid 
is  filtered  through  a  number  of  small  filters  ;  the  first  por- 
tions should  be  refiltered.  The  filtrate  is  a  greenish  fluid 
(pyocyanin)  which  by  the  careful  addition  of  acetic  or 
hydrochloric  acid  (an  excess  is  to  be  avoided)  forms  a 
voluminous  precipitate  (pyocyaneus  proteid).  This  pre- 
cipitate should  be  collected  on  a  filter,  washed  with  water, 
then  suspended  in  water  and  a  few  drops  of  a  soda  solution 
added,  when  a  dark-brown  fluid,  with  a  tendency  to  gela- 
tinize in  the  cold,  containing  about  10  per  cent,  of  the  pro- 
teid, is  obtained. 

13.254  grammes  of  the  moist  bacteria  yield  1.44  gramme 
of  dry  bacterial  substance,  and  this  alter  the  treatment 
given  above  furnishes  0.2739  gramme  of  dry  proteid  — 
19.3  per  cent.  This  proteid  leaves  11.52  per  cent,  of  ash, 
which  contains  phosphoric  acid,  but  consists  principally  of 
sodium  chloride. 

Much  smaller  amounts  of  proteid  were  obtained  from 
other  germs,  but  the  Eberth  germ,  bacillus  subtilis,  lactic 
acid  bacillus,  red  bacillus  from  potato,  and  staphylococcus 
pyogenes  aureus  furnished  considerable  quantities. 

The  chemotactic  properties  of  these  proteids  were  tested 
in  the  following  manner  :  The  dissolved  proteid  was  placed 
in  a  spindle-shaped  glass  tube,  and  the  tubes,  sterilized  by 
prolonged  boiling,  were  introduced  under  the  skin  on  the 


SUPPURATION.  131 

backs  of  rabbits  with  antiseptic  precautions,  and  the  ends 
of  the  tubes  broken  oif  subcutaneously. 

After  from  two  to  three  days  the  tubes  were  removed 
and  found  to  contain,  in  addition  to  some  of  the  proteid, 
several  millimetres  of  fibrinous  pus,  which  was  examined 
microscopically  and  by  the  preparations  of  cultures,  which 
invariably  remained  sterile.  The  proteid  of  the  Eberth 
bacillus  was  found  to  have  specially  marked  pyogenetic 
properties. 

Similar  experiments  were  made  with  the  following  crys- 
talline substances :  the  butyrate  and  valerianate  of  ammo- 
nia (each  1  per  cent,  solution),  trimethylamin  (2  per  cent.), 
ammonia  (2  per  cent.),  leucin,  tyrosin  and  glycocol  (1  per 
cent.),  urea  (5  per  cent.),  and  urate  of  ammonia  and  skatol 
(1  per  cent.).  Glycocol  and  leucin  only  were  found  to  have 
the  chemotactic  action,  and  with  these  this  action  was  but 
slight  compared  with  that  of  the  bacterial  proteids. 

The  next  experiments  were  made  with  the  object  of 
ascertaining  whether  or  not  proteids  similar  to  those  derived 
from  the  bacteria  would  cause  a  like  effect.  The  bacterial 
cellular  proteids  resemble  very  closely  vegetable  casein 
some  of  which  was  prepared  from  wheat  gluten  and  tested 
as  above.  This  proteid  was  found  to  be  possessed  of 
marked  chemotactic  properties.  The  subcutaneous  injec- 
tion of  sterilized  preparations  of  wheat-Hour  and  ground 
peas  were  also  found  to  cause  suppuration.  Negative 
results  were  obtained  with  starch  and  solutions  of  disodium 
hydric  phosphate.  From  this  it  is  concluded  that  the  active 
agent  in  the  flour  is  its  casein. 

Peptone  was  employed  Avithout  effect,  while  gelatin  was 
found  to  act  energetically.  Alkaline  albuminates  were 
prepared  from  muscle,  liver,  lungs,  and  kidney  by  treating 
finely  divided  portions  of  these  organs  with  potash  and  pro- 
ceeding as  in  the  preparation  of  the  bacterial  proteids.  All 
of  these  caused  the  formation  of  pus,  and  the  preparations 
from  the  liver  were  found  to  be  specially  potent. 

Similar  preparations  from  blood  and  egg-yolk  were 
active,  while  those  from  fibrin  and  the  white  of  egg  had  no 
effect.     Hemi-albumose  was  also  found  to  be  active,  and 


132  BACTERIAL    POISONS. 

this  fact  is  placed  in  contrast  with  the  negative  result 
obtained  with  peptone. 

One  of  the  most  interesting  results  was  obtained  by  the 
daily  injection  of  a  chemotactic  proteid  directly  into  the 
blood.  Before  the  first  injection  the  proportion  of  white  to 
red  corpuscles  was  1  :  318  ;  on  the  second  day,  1  :  126  ;  on 
the  third,  1  :  102  ;  on  the  morning  of  the  fourth,  1:73;  on 
the  afternoon  of  the  fourth,  1  :  38.  After  this  there  was 
no  further  increase.  The  absolute  number  of  red  corpuscles 
remained  unchanged,  while  the  absolute  number  of  the 
white  multiplied  sevenfold.  The  white  corpuscles  were  on 
the  first  days  often  found  in  groups  of  from  two  to  four, 
and  later,  of  from  ten  to  twenty.  This  seems  to  demon- 
strate that  these  substances  cause  an  increased  production 
of  leucocytes.  General  leucocytosis  was  induced  by  the 
similar  employment  of  vegetable  casein  and  an  alkaline 
albuminate  prepared  from  the  muscles  of  a  calf. 

Finally,  Buchnee  tested  the  action  of  this  proteid  upon 
himself.  One  cubic  centimetre  of  a  very  dilute  solution, 
containing  3.5  milligrammes  of  the  solid  proteid,  was 
injected  under  the  skin  of  the  forearm  with  antiseptic  pre- 
cautions. Two  hours  later  there  was  marked  pain  along 
the  lymphatics,  especially  localized  in  the  elbow  and  axilla. 
The  temperature  showed  no  marked  elevation  (only  37.8°). 
On  the  following  day  there  were  marked  erysipelatous 
redness  and  swelling  extending  for  some  inches  about  the 
place  of  injection,  and  accompanied  by  severe  pain.  The 
inflamed  area  felt  hot,  and  projected  distinctly  above  the 
surrounding  surface.  The  lymphatics  of  the  arm  appeared 
like  red  cords.  On  the  third  day  the  swelling  and  redness 
were  more  marked,  and  extended  from  the  wrist  to  the 
elbow.  On  the  fourth  day  the  symptoms  began  to  recede. 
Here  we  have  clinically  a  perfectly  typical  erysipelas  with 
lymphangitis,  and  Buchnee,  claims  that  all  the  cardinal 
symptoms  of  inflammation — rubor,  calor,  dolor — could  not 
be  produced  without  involvement  of  the  solid  tissues. 

Similar,  but  less  marked,  symptoms  were  induced  by  the 
injection  of  a  dilute  solution  of  vegetable  casein. 

Buchnee  states  that  bacteria  will  not  cause  inflamma- 


SUMMER    DIARRHCEAS    OF    INFANCY.  133 

tion  unless  they  be  broken  down.  The  pyogenetic  substance 
contained  within  the  bacterial  cell  can  have  no  chemotactic 
action  until  the  cell  disintegrates.  Thus,  the  anthrax 
bacillus  contains  a  pyogenetic  substance,  but  no  pus  is 
formed  in  mice  with  anthrax,  because  there  is  no  destruction 
of  the  bacilli.  This  pyogenetic  proteid  of  the  anthrax 
bacillus,  however,  manifests  its  action  in  malignant  pustule. 
These  experiments  are  of  the  greatest  interest.  We  must 
say,  however,  that  it  is  possible  that  the  bacterial  cellular 
proteid  may  be  modified  by  the  treatment  to  which  it  has 
been  subjected  in  these  experiments.  We  do  not  as  yet 
know  enough  about  the  nature  of  this  proteid  to  say  that 
its  nature  and  its  action  are  not  altered  by  being  heated  for 
hours  with  an  alkali.  However,  accepting  Buchner's 
work,  it  throws  much  light  upon  processes  which  have 
heretofore  been  but  imperfectly  understood. 

The  Summer  Diarrhceas  of  Infancy. — In  a  paper 
published  in  1888,  Vaughan  stated  that  the  microorgan- 
isms which  produce  the  catarrhal  or  mucous  diarrhoeas  of 
infancy  are  probably  only  putrefactive  or  saprophytic  in 
character,  and  that  they  prove  harmful  by  splitting  up 
complex  molecules  and  forming  chemical  poisons.  At  that 
time  it  was  generally  believed  that  a  specific  germ  would 
be  found,  but  the  truth  of  the  above  statement  is  being 
made  more  manifest  with  every  experimental  study  of  the 
subject.  Able  and  diligent  bacteriologists,  among  whom 
Booker,  in  this  country,  and  Escherich,  in  Germany, 
deserve  special  mention,  have  made  a  careful  study  of  the 
bacteria  found  in  the  intestines  and  stools  in  these  diseases, 
and  all  agree  that  no  specific  organism  has  been  found. 
Booker  has  reported  the  isolation  of  more  than  thirty 
kinds.  In  true  cholera  infantum  the  proteus  group  of  bac- 
teria was  found  in  fifteen  out  of  nineteen  cases,  but  in  the 
ordinary  diarrhoeas  there  is  no  constancy  in  the  species 
present.  Germs  which  are  frequently  found  one  year  are 
rarely  seen  in  the  cases  observed  the  next  summer.  This 
has  been  the  experience  of  all  who  have  studied  the  bacteria 
of  the  summer  diarrhoeas  of  infancy.  Vaughan  has  studied 


134  BACTERIAL    POISONS. 

the  chemical  products  of  the  germs  x,  a,  and  A  of  Booker's 
list  in  the  following  manner  and  with  the  results  as  stated 
below. 

Of  these  germs,  Booker  makes  the  following  statements  : 

"  x  was  found  almost  as  a  pure  culture  in  the  feces  of  a 
fatal  case  of  diarrhoea,  a  was  strongly  pathogenic,  when 
tested  last  winter.  A  was  isolated  last  summer ;  liquefies 
gelatin,  and  belongs  to  the  proteus  group." 

Beef-tea  cultures  of  each  of  these  germs  were  made  and 
kept  in  an  incubator  at  37°  for  forty-eight  hours.  At  the  ex- 
piration of  this  time  these  cultures  were  used  for  inoculating 
flasks  of  sterilized  beef-broth.  Eight  flasks,  each  contain- 
ing about  ten  ounces,  were  employed  for  each  germ.  These 
cultures  were  kept  in  the  incubator  at  37°  for  ten  days. 
They  were  then  twice  filtered  through  heavy  Swedish  filter- 
paper.  The  second  filtrate  was  allowed  to  fall  into  a  large 
volume  of  absolute  alcohol  feebly  acidified  with  acetic  acid. 
A  voluminous,  flocculent  precipitate  resulted  iu  each  case. 
After  the  precipitates  had  subsided  the  supernatant  fluid 
was  decanted.  The  precipitates  were  then  treated  with  dis- 
tilled water,  in  which  those  from  x  and  a  were  soluble, 
while  that  from  A  proved  insoluble.  A  large  volume  of 
absolute  alcohol  was  again  added,  and  the  mixture  allowed 
to  stand  for  four  days.  The  precipitates  from  x  and  a  com- 
pletely subsided,  leaving  the  supernatant  fluids  perfectly 
clear;  but  in  the  case  of  A  the  subsidence  was  not  com- 
plete. The  precipitates  were  collected,  by  decantation  and 
filtration,  on  porous  plates,  and  dried  over  sulphuric  acid. 
These  substances  are  proteid  in  composition,  but  differ  from 
known  proteids  and  from  one  another  That  from  x  is 
slightly  yellow,  as  seen  deposited  in  the  alcohol,  but  be- 
comes grayish  on  exposure  to  the  air.  It  is  readily  soluble 
in  water,  from  which  it  is  not  precipitated  by  heat  or  nitric 
acid,  singly  or  combined. 

It  gives  the  biuret  and  xantho-proteid  reactions.  It  is 
precipitated  by  saturating  its  aqueous  solution  with  ammo- 
nium sulphate,  and  therefore  cannot  be  classed  with  the 
peptones.    Sodium  sulphate  and  carbonic  acid  fail  to  throw 


SUMMER    DIARRHCEAS    OF    INFANCY.  135 

it  down  from  its  aqueous  solution,  consequently  we  must 
say  that  it  is  not  a  globulin. 

This  leaves  us  with  no  other  choice  than  to  place  it 
among  the  albumins,  but  we  must  admit  that  it  possesses 
properties  which  do  not  belong  to  the  known  albumins. 

The  proteid  prepared  from  cultures  of  the  germ  a  is,  as 
seen  under  the  alcohol,  very  light,  flocculent,  and  perfectly 
white,  but  so  soon  as  it  is  brought  in  contact  with  the  air 
it  begins  to  blacken,  and  finally  dries  down  on  the  porous 
plate  in  black  scales. 

It  possesses  the  same  general  properties  in  regard  to  the 
action  of  solvents  and  other  reagents  which  were  found  to 
be  possessed  by  the  proteid  obtained  from  cultures  of  x. 

The  proteid  of  A  is  peculiar,  inasmuch  as  it  is  practically 
insoluble  in  water. 

These  three  proteids  are  highly  poisonous.  When  in- 
jected under  the  skin  of  kittens  or  dogs  they  cause  vomit- 
ing and  purging,  and,  when  employed  in  sufficient  quantity, 
collapse  and  death.  Post-mortem  examination  shows  the 
small  intestine  pale  throughout  and  constricted  in  places. 
The  heart  has  been  invariably,  so  far,  found  in  diastole  and 
filled  with  blood.  The  following  brief  notes  from  the 
record  of  experiments  will  illustrate  the  nature  of  the 
symptoms  and  the  post-mortem  appearances. 

A  small  amount  of  proteid  from  bacillus  x,  dissolved  in 
water,  was  injected  under  the  skin  on  the  back  of  a  kitten 
about  eight  weeks  old.  Within  one-half  hour  the  animal 
began  to  vomit  and  purge,  aud  death  resulted  within  eigh- 
teen hours.  The  small  intestines  were  pale,  contracted  in 
places,  and  contained  a  frothy  mucus.  The  stomach  was 
distended  with  gas  and  contained  yellowish  mucus.  The 
liver  was  normal,  the  spleen  and  kidneys  congested,  and 
the  heart  distended. 

Another  kitteu  was  treated  with  the  proteid  from  bacil- 
lus a,  dissolved  in  water.  The  vomited  and  fecal  matters 
in  this  case  were  green.  The  animal  died  after  fifteen 
hours,  and  presented  appearauces  practically  identical  with 
those  mentioned  above. 

A  third  kitten  was  treated  with  some  of  the  proteid  of 


136  BACTERIAL    POISON'S. 

bacillus  A,  suspeuded  in  water,  and  presented  substantially 
the  same  symptoms  and  post-mortem  appearances. 

A  fourth  animal  was  treated  in  the  same  manner  as  the 
above  with  a  proteid  prepared  from  some  canned  meat. 
This  was  done  as  a  control  on  the  above  experiments,  and 
the  kitten  remained  unaffected.  This  experiment  demon- 
strates the  fact  that  the  poisonous  properties  are  peculiar  to 
the  bacterial  proteids. 

Concerning  the  amount  of  one  of  these  proteids  neces- 
sary to  produce  a  fatal  result  in  the  animals  experimented 
upon  a  few  experiments  have  been  made. 

Under  the  skin  on  the  back  of  a  guinea-pig,  Vaughan 
injected  ten  milligrammes  of  the  dry-scale  proteid  from 
bacillus  a.  This  caused  death  within  twelve  hours.  Of 
two  kittens  treated  with  fifteen  milligrammes  each  of  the 
a  albumin,  one  died  after  forty- eight  hours  and  the  other 
recovered  after  two  days  of  purging  and  vomiting.  Two 
dogs,  of  about  five  pounds'  weight,  had  each  forty  milli- 
grammes, and,  after  serious  illness  of  two  days'  duration, 
speedily  recovered. 

During  these  two  days  of  vomiting  and  purging  the  dogs 
were  constantly  shivering,  as  with  cold,  but  the  rectal  tem- 
perature stood  at  from  102.5°  to  103.5°  F. 

There  was  in  no  case  any  sign  of  inflammation  at  the 
point  of  injection. 

Plate  cultures  have  been  made  from  the  proteids  them- 
selves and  from  the  blood,  liver,  spleen,  and  kidneys  of 
some  of  the  animals  killed  with  the  proteid,  and  these  plates 
have  remained  sterile,  thus  demonstrating  that  no  germ 
has  been  introduced  into  the  animal  along  with  the  chem- 
ical poison. 

What  conclusions  may  we  draw  from  these  facts  when 
considered  in  connection  with  the  results  of  the  labors  of 
Booker  and  Escherich  ?  We  will  formulate  our  ideas 
in  the  following  propositions  : 

(1)  There  are  many  germs,  any  one  of  which,  when  in- 
troduced into  the  intestines  of  the  infant,  under  certain 
favorable  conditions,  may  produce  diarrhcea. 

As  has  been   stated,  many  different  germs  have  been 


SUMMER    DIAEEHCEAS    OF    INFANCY.  137 

found  in  the  intestines  of  infants  suffering  from  summer 
diarrhoea,  and  we  now  find  that  three  species  of  these  are 
capable  of  producing  chemical  poisons,  which  induce  effects 
substantially  identical  with  the  symptoms  observed  in  the 
infants,  and  it  is  not  unreasonable  to  suppose  that  many 
other  of  these  germs  produce  similar  poisons. 

(2)  Many  of  these  germs  are  probably  truly  saprophytic. 
A  germ  growing  in  the  intestine  does   not  necessarily 

feed  upon  living  tissue.  The  food  in  the  duodenum  before 
absorption  has  no  more  vitality  than  the  same  material  in 
the  flask.  Moreover,  the  excretions  poured  into  the  intes- 
tines from  the  body  are  not  supposed  to  be  possessed  of 
vitality.  A  germ  which  will  grow  upon  a  certain  medium 
in  the  flask  and  produce  a  poison  will  grow  on  the  same 
medium  in  the  intestine  and  produce  the  same  poison,  pro- 
vided it  is  not  destroyed  by  some  secretion  of  the  body. 

(3)  The  only  digestive  secretion  which  is  known  to  have 
auy  decided  germicidal  effect  is  the  gastric  juice;  therefore, 
if  the  secretion  be  impaired  there  is  at  least  the  possibility 
that  the  living  germ  will  pass  on  to  the  intestine,  will  there 
multiply,  aud  will,  if  it  be  capable  of  so  doing,  elaborate  a 
chemical  poison  which  may  be  absorbed. 

There  is  no  longer  any  doubt  that  the  acid  of  the  gastric 
juice  has  a  marked  germicidal  effect  upon  many  of  the 
microorganisms. 

Vaughan  has  found  that  an  exposure  to  a  two-tenths 
per  cent,  solution  of  hydrochloric  acid  for  half  an  hour  will 
destroy  Eberth's  germ  and  two  poison-producing  bacilli 
which  he  has  isolated  from  drinking-water  which  was 
believed  to  have  caused  typhoid  fever.  Although  the  ger- 
micidal effect  of  this  acid  has  not  been  tried  on  the  bacteria 
under  consideration,  doubtless  it  will  be  found  to  be  con- 
siderable. 

The  chief  reason  why  the  breast-fed  child  has  a  better 
chance  for  life  than  the  one  fed  upon  cow's  milk  lies  in  the 
fact  that  the  former  gets  its  food  germ-free  ;  but  a  second 
reason  is  to  be  found  in  the  larger  amount  of  acid  required 
to  neutralize  the  cow's  milk,  as  has  been  pointed  out  by 


138  BACTERIAL    POISONS. 

Escherich.     The  gastric  juice  is  the  physiological  guard 
against  infection  by  way  of  the  intestines.1 

It  is  also  possible  that  some  of  the  secretions  poured  into 
the  intestines  have  germicidal  properties,  or  that  the  cells, 
in  absorbing  the  poisonous  proteids,  may  to  a  limited  ex- 
tent so  alter  them  that  they  are  no  longer  poisonous,  or 
that  in  a  perfectly  normal  condition  the  liver  may  be  able 
to  prevent  these  poisons  from  entering  the  general  circula- 
tion without  change.  These  are  all  possibilities,  which 
science  at  some  time  in  the  future  will  investigate. 

(4)  Any  germ  which  is  capable  of  growing  and  produc- 
ing an  absorbable  poison  in  the  intestine  is  a  pathogenic 
germ. 

It  is  not  necessary  that  a  germ  be  capable  of  growing 
and  causing  disease  and  death  when  injected  under  the  skin 
or  into  the  blood  in  order  to  establish  its  right  to  rank  with 
the  pathogenic  germs.  In  the  blood  the  organism  is  acted 
upon  by  a  wholly  different  fluid  from  that  with  which  it 
is  surrounded  in  the  intestine,  and  the  germicidal  properties 
of  the  blood  have  been  unquestionably  demonstrated. 

(5)  The  proper  classification  of  germs  in  regard  to  their 
relation  to  disease  cannot  be  made  from  their  morphology 
alone,  but  must  depend  largely  upon  the  products  of  their 
growth. 

As  has  been  stated,  three  microorganisms,  differing  suf- 
ficiently to  be  recognized  as  of  different  species,  produce 
poisons,  all  of  which  induce  vomiting  and  purging,  and, 
when  used  in  sufficient  quantity,  death.  Morphologically 
these  bacilli  may  not  be  closely  related,  but  physiologically 
they  are  near  akin. 

If  these  deductions  be  true,  we  will  try  to  avoid  the 
introduction  into  the  alimentary  canal,  not  only  of  the  so- 
called  specific  pathogenic  germs,  but  of  all  toxicogenic 
microorganisms. 

1  It  has  been  said  that  this  statement  cannot  be  true,  because  there  are 
other  acids  which  are  more  powerful  germicides  than  hydrochloric  acid, 
but  there  is  no  force  in  this  argument.  The  question  is  not  whether  the 
stomach  is  supplied  with  the  very  best  germicide,  but  whether  it  is  sup- 
plied with  any  at  a'l.  The  human  eye  may  not  be  a  perfect  mechanism, 
but  it  is  man's  only  organ  of  vision. 


TYPHOID    FEVER.  139 

Baginsky  and  Stadthagen  have  obtained  from  cul- 
tures of  the  "  white  liquefying  bacterium  "  of  the  former  a 
poisonous  proteid  which  produces  in  mice,  after  about  five 
hours,  slight  dyspuoea.  The  coat  becomes  rough,  the  ani- 
mal sits  with  drooping  head,  and  when  forced  to  move  does 
so  sluggishly,  but  without  any  evidence  of  paralysis.  The 
marked  apathy  increases,  and  death  results  after  two  or 
three  days.  Section  shows  an  infiltration  about  the  place 
of  injection,  congestion  of  the  spleen,  liver,  and  peritoneum. 
The  iutestine  is  hypersemic  throughout  its  entire  length, 
and  its  upper  portion  contains  a  reddish-brown  fluid. 

From  cultures  of  the  same  bacterium  Baginsky  and 
Stadthagen  have  also  obtained  a  poisonous  ptomaine, 
which  is  probably  identical  with  one  found  by  Brieger 
in  putrid  horseflesh,  and  which  has  the  formula  C7H]7N02. 

That  tyrotoxicon  is  one  of  the  causes  of  the  violent 
choleraic  diarrhoea  of  children  there  can  scarcely  be  a 
doubt.  The  symptoms  induced  by  the  poison  canuot  be 
distinguished  from  those  of  the  disease.  The  post-mortem 
appearances  are  very  much  alike,  if  not  identical,  and  the 
poison  has  been  found  in  a  sample  of  milk  a  part  of  which 
had  been  given  to  a  child  not  more  than  two  hours  before 
the  first  symptoms  of  a  violent  attack  of  the  disease  made 
themselves  manifest. 

Typhoid  Fever.  —  In  1880,  Eberth  discovered  a 
bacillus  which  he  believed  to  be  the  cause  of  typhoid  fever, 
and  this  belief  has  been  quite  generally  accepted.  In  the 
first  edition  of  this  work  it  was  stated  that  the  fever  and 
the  characteristic  lesions  of  the  disease  had  been  produced 
in  animals  by  inoculation  with  this  germ.  This  is  now 
known  to  be  erroneous.  As  has  been  stated  (page  93),  the 
essential  lesions  of  typhoid  fever  may  be  produced  in  ani- 
mals with  a  number  of  microorganisms,  among  which, 
however,  the  Eberth  bacillus  is  not  included.  The  results 
obtained  by  Frankel  and  Simmonds,  and  Seitz  have  been 
shown  by  Beumer  and  Peiper  to  be  fallacious,  and  the 
germ  with  which  the  experiments  were  made  by  Yaughan 
and  Now,  and  mentioned  in  the  first  edition,  is  known 


140  BACTERIAL    POISONS. 

not  to  be  identical  with  that  of  Eberth.  It  is  true  that 
this  germ  induced  in  dogs  a  continued  fever  of  from 
twenty-eight  to  thirty-five  days  in  duration,  terminating 
in  some  instances  fatally  and  revealing  ulceration  and  per- 
foration of  the  small  intestines,  but  for  this  reason  it  is 
known  to  be  different  from  Eberth's  bacillus,  because  the 
latter  never  induces  these  effects.  Notwithstanding  this 
failure  to  affect  the  lower  animals,  the  majority  of  bacteri- 
ologists believe,  as  has  been  stated,  that  the  Eberth 
bacillus  is  the  sole  and  only  cause  of  typhoid  fever.  In 
this  believe  Vaughan  refuses  to  coucur,  and  claims  that 
the  Eberth  bacillus  as  found  in  the  spleen  after  death  is 
an  involution  form  of  any  one  of  a  number  of  germs 
which  are  found  in  certain  waters.  As  this  is  not  the 
place  for  an  extended  discussion  of  purely  morphological 
questions,  the  reader  is  referred  to  the  literature  of  the 
subject,  and  we  will  content  ourselves  with  giving  the 
following  summary  of  what  is  known  concerning  the 
chemical  products  of  the  Eberth  bacillus  and  of  the 
germs  studied  by  Vaughan. 

In  1885,  Brieger  obtained  from  pure  cultures  of  the 
Eberth  bacillus  a  poisonous  ptomaine,  which  produced  in 
guinea-pigs  a  slight  flow  of  saliva,  frequency  of  respira- 
tion, dilatation  of  the  pupils,  profuse  diarrhoea,  paralysis, 
and  death  within  from  twenty-four  to  forty-eight  hours. 
Post-mortem  examination  showed  the  heart  in  systole,  the 
lungs  hypersemic,  and  the  intestines  contracted  and  pale. 
At  first  Brieger  was  inclined  to  regard  this  as  the  specific 
poison  of  typhoid  fever  and  named  it  typhotoxine.  How- 
ever, he  has  more  recently  modified  his  opinion  and  is 
inclined  to  regard  typhoid  fever  as  due  to  a  mixed  in- 
fection. 

Brieger  and  Frankel  have  found  in  cultures  of  the 
Eberth  bacillus  a  proteid  which  causes  death  in  rabbits 
after  from  eight  to  ten  days.  They  say  nothing  about  the 
symptoms. 

In  1889,  Vaughan  isolated  from  mixed  cultures  from 
typhoid  stools  a  base,  forming  crystalline  salts  and  capable 
of  inducing  in  cats  and  dogs  a  marked  elevation  of  tern- 


TYPHOID    FEVER.  141 

perature  accompanied  by  severe  purging.  The  following 
is  the  record  of  one  experiment  with  this  substance : 
"  An  aqueous  solution  of  the  crystals  was  given  to  a  dog 
by  the  mouth  at  3  p.m.  The  rectal  temperature  before  the 
administration  was  101°  F.  At  3.15,  retching  and  vomit- 
ing set  in  and  continued  at  intervals  for  more  than  two 
hours.  At  3.30,  the  temperature  was  103°  F.  At  3.55, 
the  animal  began  to  purge.  The  first  discharges  contained 
much  fecal  matter,  but  subsequently  they  were  watery 
and  contained  mucus  plainly  stained  with  blood.  At  4, 
the  temperature  was  1U3.50  F.  and  remained  the  same  at 
4.30.  The  animal  was  not  seen  again  until  10  a.m.  the 
next  day,  when  its  temperature  was  100.5°,  and  recovery 
seemed  complete." 

This  base  was  not  obtained  in  quantity  sufficient  for  an 
ultimate  analysis.  The  platino-chloride  crystallizes  in  fine 
rhombic  prisms  and  the  hydrochloride  in  long,  delicate, 
red  needles.  The  red  color  seems  to  be  inherent  to  the 
substance  and  not  due  to  impurities.  The  mercury  and 
platinum  compounds  are  insoluble  in  alcohol,  soluble  in 
water.  The  hydrochloride  is  soluble  in  both  water  and 
alcohol. 

In  1890,  Vaughan  reported  the  isolation,  from  water 
supposed  to  cause  typhoid  fever,  of  a  number  of  toxi- 
cogenic  germs.  The  chemical  products  of  two  of  these 
have  been  studied.  They  belong  to  the  proteids,  and  an 
analysis  of  one  of  them  by  Freer  shows  it  to  belong  to  the 
nucleins.  These  poisons  are  soluble  in  water,  the  opales- 
cent solution  showing  a  distinctly  acid  reaction.  They  are 
not  precipitated  by  heat  or  nitric  acid  singly  or  combined. 
They  dissolve  in  nitric  acid,  forming  a  colorless  solution, 
which  becomes  yellow  on  the  addition  of  ammonia.  They 
dissolve  in  caustic  alkalies  and  the  solution  becomes  purple 
on  the  addition  of  a  dilute  solution  of  copper  sulphate. 

On  white  rats  these  poisons  produce  symptoms  which  are 
identical  with  those  which  follow  inoculations  with  the 
living  germs.  The  rat  seems  to  shiver  with  cold  and  gives 
evidence  of  abdominal  pain.  It  lies  with  its  limbs  flexed 
and  head  drawn  down  for  a  few  seconds,  then  stretches  out 


142  BACTERIAL    POISONS. 

the  limbs.  It  lies  on  the  side  for  a  short  time,  then  sits 
with  the  head  drawn  under  the  body. 

Dogs  shiver  as  with  cold,  but  at  the  same  time  the  rectal 
temperature  is  from  one  to  four  degrees  above  the  normal. 
In  some  instances  vomiting  and  purging  have  been  induced. 

The  following  experiments  seem  to  show  that  the  poison 
accumulates  in  the  nerve-centres  : 

Two  guinea-pigs  were  treated  with  hypodermic  injec- 
tions of  one  of  these  poisons,  the  amount  used  being  about 
ten  times  the  dose  which  ordinarily  proves  fatal  to  these 
animals.  Within  twelve  hours  both  were  dead.  Plate 
cultures  made  from  the  liver,  spleen,  blood,  brain,  and 
spinal  cord  remained  sterile.  Small  quantities  of  the  brain 
and  cord  were  rubbed  up  iu  a  sterilized  dish  with  sterilized 
water,  and  two  c.  c.  of  the  emulsion  were  injected  under 
the  skin  of  each  of  four  guinea-pigs.  These  animals  seemed 
to  be  very  excitable  the  next  day,  throwing  themselves 
about  violently  in  the  cages  when  slight  noises  were  made 
near  them.  Within  a  period  of  from  sixteen  to  twenty- 
four  days  all  died.  This  experiment  needs  repetition,  and 
it  will  be  necessary  to  prepare  and  inject  similar  emulsions 
made  from  other  organs  before  any  positive  conclusions  can 
be  drawn. 

In  a  study  of  fatal  cases  of  typhoid  fever  at  Bucharest 
Babes  finds  that  the  typical  germ  differs  markedly  from 
that  of  Eberth. 

Swine-plague,  or  Hog-cholera. — The  researches  of 
Loffler,  Schutz,  Lydtin,  and  Schottelius  in  Europe, 
and  of  Billings  and  Salmon  in  this  country,  have  demon- 
strated the  existence  among  swine  of  at  least  three  infectious 
diseases.     These  are — 

(1)  Hog-erysipelas,  or  rouget  of  France,  or  Schweine- 
rothlauf  of  Germany. 

(2)  German  swine-plague,  or  Schweineseuche. 

(3)  American  swine-plague  (Billings),  or  hog- cholera 
(Salmon). 

The  first  two  of  these  are  exclusively  European  diseases, 
and  their  chemical  poisons  have  not  been  studied. 


SWINE-PLAGUE.  143 

The  American  swine-plague  is  preeminently  a  disease  of 
the  digestive  tract  involving  most  markedly  the  large  in- 
testine. It  is  the  great  swine  disease  of  this  country,  and 
is  probably  present  in  England,  where  it  is  associated  with 
other  diseases  under  the  name  of  swine-fever.  A  disease 
which  was  observed  in  Denmark  and  Sweden  for  the  first 
time  in  1888-89  and  known  as  swine-pest  or  swine-diph- 
theria, has  been  shown  by  Selandee,  Feosch,  and  others 
to  be  identical  with  our  swine-plague.  In  the  summer  of 
1889  France  was  visited  by  a  swine  disease,  which  is  con- 
sidered by  Coenil  and  Chantemesse  to  be  identical  with 
the  German  swine-plague,  but  which  Rietsch  and  Jobeet, 
after  a  comparative  study  of  the  microorganisms,  pronouuce 
as  the  American  disease.  In  this  country  we  have  at  pres- 
ent no  positive  demonstration  of  the  existence  of  any  other 
infectious  swine  disease.  The  swine-plague  of  Salmon 
has  been  the  subject  of  considerable  discussion,  but  its  ex- 
istence can  hardly  be  said  to  be  established. 

The  following  statements  concerning  the  chemical  poisons 
refer  to  the  swine-plague  of  Billings  or  the  hog-cholera 
of  Salmon,  which  are  only  two  names  for  one  disease. 

In  pure  cultures  of  this  bacillus  Novy  has  found  a  poi- 
sonous base,  which  probably  has  the  composition  C10H26N2, 
and  to  which  he  has  provisionally  given  the  name,  suso- 
toxine.  One  hundred  milligrammes  of  the  hydrochloride 
of  this  base  causes  in  white  rats  convulsive  tremors  and 
death  within  one  and  one-half  hours.  Post-mortem  exam- 
ination shows  the  heart  in  diastole,  lungs  pale,  stomach 
contracted,  a  serous  effusion  in  the  thoracic  cavity,  and  the 
subcutaneous  tissue  pale  and  cedematous. 

Novy  has  also  obtained  a  poisonous  proteid  from  cul- 
tures of  this  germ.  The  following  experiments  illustrate 
the  effects  obtained  with  this  body  :  100,  50,  and  25  milli- 
grammes, respectively,  were  injected  into  three  young  rats 
from  the  same  litter.  The  animal  which  received  100  mg. 
soon  began  to  crawl  about  on  its  belly,  being  unable  to  rise. 
The  eyes  were  soon  filled  with  a  thick  secretion  and  the 
toes  became  red.  Finally  it  became  quiet,  lying  on  its 
belly,  with  feet  extended.     The  respirations  became  deeper, 


144  BACTEKIAL    POISONS. 

and  a  coma-like  condition  set  in.  The  animal  died,  with- 
out convulsions,  within  about  three  hours.  The  rat  which 
received  50  nag.  went  through  the  same  course  of  symptoms, 
but  these  were  less  intense.  Death  resulted  four  hours 
after  the  injection.  The  one  which  received  25  mg.  be- 
came very  sick,  but  finally  recovered,  and  one  week  later 
it  was  given  another  injection  of  30  mg.,  which  produced 
scarcely  any  effect.  Then  it  was  treated  at  intervals  of 
five,  three,  five,  two,  and  four  days,  respectively,  to  40,  50, 
75,  100,  and  125  mg.  without  effect.  Three  days  after  the 
last  injection  the  animal  was  inoculated  with  one  c.  c.  of  a 
bouillon  culture  of  the  highly  virulent  germ.  Only  a 
slight  temporary  effect  was  observed  during  the  first  day, 
after  which  recovery  was  complete  and  permanent.  A 
control  rat  which  was  given  the  same  quantity  of  the  cul- 
ture sickened  the  next  day  and  died  one  week  later.  From 
this  it  will  be  seen  that  the  animal  was  rendered  immune 
against  the  disease. 

Schweinitz  also  reports  the  detection  of  a  slightly 
poisonous  base,  which  he  designates  as  sucholotoxin,  and  a 
poisonous  proteid,  and  with  these  he  has  been  able  to  secure 
immunity  in  guinea-pigs  against  the  virulent  germ.  The 
proteid  body  is  classed  among  the  albumoses,  and  is  said 
to  crystallize  in  white,  translucent  plates  when  dried  in 
vacuo  over  sulphuric  acid  and  to  form  needle-like  crystals 
with  platinum  chloride.  No  one  else  has  reported  a  crys- 
talline bacterial  proteid,  and  this  body  is  deserving  of  a 
more  extended  study. 

Rabbit  Septicemia. — Hoffa  has  killed  rabbits  by 
inoculation  with  pure  cultures  of  the  bacillus  of  this  disease, 
and  has  isolated  from  the  bodies  of  these  animals  methyl- 
guanidin,  while  in  the  bodies  of  healthy  rabbits  this  poison 
could  not  be  found.  The  fatal  dose  of  methylguanidin  for 
rabbits  was  found  to  be  0.2  gramme  when  given  subcu- 
taneously.  Since  Hueppe  has  suggested  that  the  bacte- 
rium of  chicken-cholera  is  identical  with  that  of  rabbit 
septicaemia,  chickens  were  poisoned  with  methylguanidin, 


PUERPERAL    FEVER.  145 

and  the  symptoms  were  observed  to  be  analogous  to  those 
of  the  disease. 

Pneumonia. — Bonardi  has  made  a  chemical  study  of 
the  diplococcus  of  Frankel.  He  finds  certain  poisons — 
ptomaines — which  he  has  been  unable  as  yet  to  obtain  in 
quantity  sufficient  for  ultimate  analysis.  He  also  claims 
to  have  secured  immunity  against  the  germ  by  treating 
rabbits  with  small  quantities  of  the  chemical  poisons. 

Malignant  (Edema. — Kerry  finds  that  the  bacillus 
of  this  disease  decomposes  albumin  with  the  formation  of 
fatty  acids,  leucin,  hydro-paracumaric  acid,  and  a  foul- 
smelling  oil  of  the  composition  C8H1604.  This  oil  is  in- 
soluble in  water,  alkalies,  and  acids,  easily  soluble  in  ether, 
benzol,  bisulphide  of  carbon,  and  alcohol.  It  is  optically 
inactive,  and  on  being  oxidized  furnishes  valerianic  acid. 
Nothing  is  said  concerning  its  action  upon  animals. 
Among  the  gaseous  products  are  carbonic  acid,  hydrogen, 
and  marsh  gas.  The  author  was  unable  to  determine 
whether  or  not  free  nitrogen  is  formed. 

Puerperal  Fever. — Bourget  claims  to  have  isolated 
several  ptomaines  from  the  urine  of  women  with  puerperal 
fever.  His  conclusions  are  as  follows:  (1)  In  puerperal 
fever  the  urine  contains  highly  poisonous  bases.  (2)  The 
toxicity  of  the  urine  is  most  marked  when  the  symptoms 
of  the  disease  are  most  grave,  and  diminishes  as  the  symp- 
toms abate.  (3)  The  ptomaines  obtained  from  the  urine 
prove  fatal  when  injected  into  frogs  and  guinea-pigs.  (4) 
Toxic  bases,  resembling  those  obtained  from  the  urine,  were 
extracted  from  the  viscera  of  a  woman  who  had  died  of 
puerperal  fever. 


CHAPTER    VI. 

THE    NATURE    OF    IMMUNITY-GIVING   SUBSTANCES. 

Ogata  aud  Jasuhara  find  that  anthrax  bacilli  grown 
in  the  blood-sernm  of  animals  naturally  immune  to  the 
disease  will  not  on  subsequent  inoculation  induce  the  dis- 
ease in  animals  naturally  susceptible.  Thus,  anthrax 
germs  grown  in  frog-blood  make  mice  sick,  but  do  not 
prove  fatal  to  them,  aud  those  grown  on  the  blood-serum 
of  white  rats  or  dogs  have  a  similar  effect  upon  rabbits; 
but  germs  grown  in  the  blood  of  auimals  not  immune  kill 
both  mice  and  rabbits.  They  also  find  that  the  injection 
of  one  drop  of  frog  blood-sernm  or  one-half  drop  of  serum 
from  a  dog  into  a  mouse,  any  time  within  seventy-two 
hours  before  to  five  hours  after  inoculation  with  anthrax, 
protects  this  animal  from  the  disease.  A  guinea-pig  weigh- 
ing 400  grammes  was  given  tweuty  drops  of  frog's  blood 
diluted  with  the  0.6  per  cent,  salt  solution  and  immediately 
thereafter  inoculated  with  virulent  anthrax ;  the  animal 
became  slightly  sick,  but  soon  recovered.  The  same  was 
true  of  a  rabbit  weighing  1500  grammes  which  was  treated 
with  8  c.c.  of  defibrinated  dog's  blood.  The  experimenters 
conclude  that  one-fourth  of  a  drop  of  the  serum  of  the  dog 
diluted  to  three  times  its  volume  with  the  salt  solution  is 
the  smallest  amount  which  will  give  immunity  against 
anthrax  to  a  mouse  of  10  grammes. 

Kitasato  and  Behring  have  secured  immunity  in 
some  animals  against  tetanus  aud  diphtheria  by  the  follow- 
ing methods  : 

(1)  By  the  method  of  Frankel  (for  diphtheria),  which 
has  been  given.     (See  page  128.) 

(2)  By  the  addition  of  iodine  trichloride  to  cultures  four 
weeks  old,  in  the  proportion  of  1  :  500 ;  allow  to  stand  for 
sixteen  hours ;  inject  2  c.c.  into  the  abdominal   cavity  of  a 


IMMUNITY-GIVING-    SUBSTANCES.  147 

guinea-pig ;  three  weeks  later  inject  0.2  c.c.  of  a  culture 
in  bouillon  containing  iodine  trichloride  in  the  proportion 
of  1  :  5500. 

(3)  By  the  metabolic  products  of  the  diphtheria  bacillus 
in  the  living  body.  In  the  pleural  cavities  of  guinea-pigs 
killed  by  inoculation  with  the  germ  there  is  often  a  reddish, 
germ-free  transudate;  10  c.c.  to  15  c.c.  of  this  kills  guinea- 
pigs;  small  amounts  give  immunity. 

(4)  By  inoculating  with  the  virulent  germ  and  arresting 
the  growth  of  the  same  with  iodine  trichloride,  gold-sodium 
chloride,  naphthylamine,  or  carbolic  acid.  Of  eight  guinea- 
pigs,  each  of  which  was  inoculated  with  0.3  c.c.  of  a  viru- 
lent culture,  two,  which  were  not  treated,  died  within 
twenty-four  hours  ;  four,  which  had — two  each — a  1  per 
cent,  and  a  2  per  cent,  solution  of  iodine  trichloride  injected 
immediately  and  at  the  place  of  inoculation,  recovered  ;  of 
two  which  had  the  same  treatment  six  hours  after  the 
inoculation,  one  died  after  four  days. 

(5)  By  peroxide  of  hydrogen  in  diluted  sulphuric  acid. 
Guinea-pigs  bear  from  1  :  4000  to  1  :  2500  ;  mice,  1  :  2000 
to  1  :  800 ;  rabbits,  less  than  1  :  1500  of  this  substance 
per  body-weight.  Injections  of  this  solution  before  inocu- 
lation give  more  or  less  immunity,  or,  rather,  increase  the 
resistance  to  the  disease ;  given  after  inoculation  it  hastens 
death. 

None  of  these  methods  are  applicable  to  the  prevention 
or  treatment  of  the  disease  in  man. 

Tizzoni  and  Cattani  have  reviewed  the  above  state- 
ments in  so  far  as  they  refer  to  tetanus.  These  experi- 
menters find  that  the  addition  of  an  equal  volume  of 
either  a  2  per  cent,  solution  of  fresh  chlorine  water  or 
iodine  trichloride,  or  a  5  percent,  solution  of  phenylic  acid 
to  the  poisonous,  filtered  tetanus  culture  destroys  the  tox- 
icity of  the  same;  but  they  state  that  the  injection  of  these 
substances  into  animals  either  before  or  after  inoculation 
with  the  germ  has  no  effect  upon  the  development  or  course 
of  the  disease. 

However,  they  do  find  that  the  blood-serum  of  an  ani- 
mal   which    is    immune    will    protect    against    either    the 


148  BACTERIAL    POISONS. 

living  germ  or  the  germ-free  culture.  Pigeons  and  dogs 
are  but  slightly  susceptible  to  tetanus  and  they  are  made 
still  less  so  by  being  treated  for  a  number  of  times  with 
small  quantities  of  the  virulent  culture.  After  each  re- 
covery these  animals  are  found  to  be  less  susceptible,  and 
finally  they  acquire  a  high  degree  of  immunity,  and  then 
their  blood  is  employed  in  securing  immunity  in  other 
animals  much  after  the  manner  already  detailed  for 
anthrax. 

Tizzoni  and  Cattani  have  attempted  to  ascertain  the 
nature  of  that  constituent  of  the  blood-serum  which  gives 
immunity.  In  these  experiments  serum  from  a  dog  which 
had  been  rendered  immune  against  tetanus  was  employed. 
In  the  first  place,  a  filtered  culture  of  the  tetanus  germ 
was  concentrated  in  vacuo  at  40°  until  one-half  c.c.  of 
it  would  kill  a  rabbit  within  thirty-six  hours.  To  this 
amount  of  the  culture,  the  blood-serum  was  added  after 
having  been  subjected  to  varied  treatment,  and  the  whole 
was  subsequently  injected  into  a  rabbit.  The  blood-serum 
retains  its  antitoxic  properties  when  kept  in  the  dark  at 
15°  for  some  days,  and  it  may  be  heated  to  60°  without 
injury.  A  temperature  of  65°  weakens,  and  one  of  68° 
(the  temperature  at  which  the  serum  coagulates)  completely 
destroys  the  antitoxic  properties  of  the  serum.  The 
"  tetanus-antitoxine"  is  non-diffusible.  It  is  precipitated 
from  the  blood-serum  on  the  addition  of  absolute  alcohol 
and  from  the  dried  percipitate  it  may  be  extracted  either 
with  water  or  glycerin,  though  very  slowly  with  the 
latter.  From  these  facts  it  is  concluded  that  the  antitoxin 
is  a  proteid  with  the  characteristics  of  an  enzyme. 

Hankin  gives  the  following  argument  in  favor  of  the 
theory  that  immunity  is  not  due  to  ptomaines,  but  to  pro- 
teids  :  "  It  is  generally  admitted  that  in  acquired  immunity 
against  a  disease  we  are  dealing  (for  the  most  part,  at  least) 
with  a  phenomenon  of  the  nature  of  acquired  tolerance  of 
a  poison.  If  we  consider  what  this  theory  really  implies, 
and,  further,  suppose  that  the  poison  involved  is  a  pto- 
maine or  other  body  of  an  alkaloidal  nature,  numerous 
diffculties  immediately  present   themselves,     For,  in  the 


IMMUNITY-GIVING    SUBSTANCES.  149 

first  place,  if  acquired  immunity  is  of  this  nature,  we  are 
dealing  with  an  acquired  tolerance  of  a  poison,  which 
tolerance  is  conferred  by  administering  a  single  dose,  or  at 
most  a  very  limited  number  of  closes.  Further,  this  ac- 
quired tolerance,  thus  easily  obtained,  is  very  permanent, 
lasting  for  months,  or  even  years.  Now,  though  acquired 
tolerance  of  alkaloids  is  constantly  observed,  it  is  but 
limited  in  degree,  aud  only  obtained  as  the  result  of  a 
long-continued  succession  of  doses.1  Secondly,  since  ac- 
quired tolerance  of  this  hypothetical  poison  results  in  the 
microbe  being  no  longer  capable  of  living  in  the  body, 
this  theory  implies  that  the  poison  in  question  is  one  that 
is  produced  by  the  microbe  in  order  to  live  there.  In 
other  words,  that  it  is  a  poison  capable  of  lowering  the 
bacteria-killing  power  possessed  by  every  living  animal 
body.2 

"  Of  course,  it  is  conceivable  that  a  ptomaine  might  be 
concerned  in  doing  this,  but,  so  far  as  I  know,  no  parallel 
to  such  action  can  be  found  among  bodies  of  an  alkaloidal 
nature. 

"  When,  however,  we  turn  to  what  is  known  of  poison- 
ous proteids,  we  at  once  find  that  they  have  properties 
analogous  to  those  of  the  hypothetical  immunity-giving 
poison. 

"  First,  as  regards  the  question  of  tolerance :  Two  poisons 
are  known,  which,  in  the  nature  of  the  tolerance  they  pro- 
duce, resemble  the  hypothetical  poison  in  question.  Both 
of  them  are  albumoses.  The  first  is  the  ordinary  hemi- 
albumose  of  proteid  digestion.  It  is  known  that  the  injec- 
tion of  a  single  minute  dose  confers  immunity  against  a 

1  Carbone  claims  to  have  obtained  immunity  in  rabbits  against  the  action 
of  the  proteus  vulgaris  by  means  of  not  more  than  two  previous  injections 
of  small  quantities  of  neurin  obtained  from  cultures  of  the  proteus.  He 
still  further  states  that  immunity  against  the  same  germ  is  obtained  by 
muscarin,  which  produces  physiological  effects  practically  identical  with 
those  of  this  neurin. 

2  With  this  statement  we  must  take  issue.  The  experiments  already 
given  in  which  immunity  is  induced  in  a  susceptible  animal  by  the  injection 
ot  the  serum  of  the  blood  of  an  animal  naturally  immune  show  that  the 
immunity-giving  substance  is  not  necessarily  of  bacterial  origin,  and  cer- 
tainly that  it  is  not  necessarily  a  product  of  the  germ  against  which  the 
immunity  is  secured. 


150  BACTERIAL    POISONS. 

further  dose  for  a  period  of  twelve  hours.  The  second 
albumose  is  the  poisouous  principle  of  snake-poison. 
Sew  all,  in  1887,  published  a  very  interesting  research  on 
acquired  immunity  against  snake-poison.  He  showed  that 
it  was  possible,  by  the  injection  of  a  few  minute  doses,  to 
give  pigeous  such  a  tolerance  of  this  substance  that,  three 
months  after  the  treatment,  they  were  able  to  stand  what 
would  otherwise  be  seven  times  the  lethal  dose.  He  sug- 
gests in  his  paper  that,  by  inoculation  with  the  ptomaines 
produced  by  bacteria,  it  may  be  possible  to  protect  animals 
against  their  disease-producing  powers,  although  the  re- 
markable case  of  tolerance  he  had  discovered  suggested  that 
not  ptoma'iues,  but  albumoses,  were  the  substances  con- 
cerned in  giving  immunity  against  a  disease;1  for  I  sug- 
gest that  this  fact — that  the  only  cases  of  tolerance  known 
which  resemble  the  tolerance  implied  in  disease-immunity 
are  cases  of  tolerance  against  albumoses — strongly  suggests 
that  immunity  against  a  disease  is  immunity  against  an 
albumose  produced  by  the  microbe." 

In  conformity  with  the  above-stated  theory,  Hankin 
prepared,  as  we  have  already  stated,  from  cultures  of  the 
anthrax  bacillus  a  poisonous  albumose,  which,  when  em- 
ployed in  small  doses,  gives  immunity;  in  large  doses, 
proves  fatal.  Hankin  endeavored  to  separate  any  ferment 
that  might  be  present  and  to  which  the  immunity  might 
possibly  be  due.  A  quantity  of  lime  water  was  added  to 
a  solution  of  the  albumose  and  the  lime  precipitated  by  the 
addition  of  phosphoric  acid.  Theoretically,  the  precipitate 
should  contain  any  ferment  present,  and  the  immunity- 
giving  property  of  the  albumose  would  be  diminished  by 
the  amount  of  the  ferment  thus  removed,  in  case  the  im- 
munity be  due  to  the  ferment.  However,  the  albumose 
was  found  to  have  lost  none  of  its  immunity-producing 
power.  From  this  Hankin  concludes  that  the  albumose 
is  the  real  immunity-producing  agent.     He  does  not  in- 

1  We  would  suggest  the  fact  that  in  1887  it  was  not  known  that  bacteria 
produce  albumoses,  and  at  that  time  the'  term  "ptomaine"  was  employed 
to  indicate  all  the  bacterial  poisons. 


DEVELOPMENT    OF    INFECTIOUS    DISEASES.      151 

form  us  whether  or  not  any  test  of  the  phosphate  precipi- 
tate was  made. 

Bacterial  Products  which  Favor  the  Develop- 
ment of  Infectious  Diseases. — Roger  has  made  a 
very  interesting  contribution  on  this  subject,  and  if  his 
work  be  confirmed  the  question  of  mixed  infection  will 
become  more  important  than  it  has  been  supposed  to  be. 
Rabbits  are  not  naturally  susceptible  to  the  germ  of  char- 
bon  symptomatique  ;  indeed,  inoculation  with  pure  cultures 
of  the  bacillus  has  no  visible  effect.  But  Roger  finds  that 
if  the  staphylococcus  pyogenes  aureus,  proteus  vulgaris,  or 
bacillus  prodigiosus  be  injected  into  the  animal  at  the  same 
time  with  the  germ  of  charbon  symptomatique  the  latter 
develops  and  produces  the  disease.  The  same  result  is 
obtained  when  a  sterilized  culture  of  the  bacillus  prodigi- 
osus is  employed.  He  at  first  supposed  that  the  chemical 
products  of  the  bacillus  prodigiosus  so  lowered  the  vitality 
of  the  tissues  that  the  pathogenic  germ  was  enabled  to 
establish  itself;  but  he  found  that  the  same  results  were 
obtained  when  the  two  inoculations  were  made  in  distant 
parts  of  the  body.  The  most  marked  effects  were  seen 
when  the  sterilized  culture  was  injected  into  a  vein  and  the 
charbon  bacillus  subcutaneously.  In  these  instances  the 
rabbits  rapidly  developed  enormous  tumors,  and  died 
within  twenty-four  hours.  One  drop  of  the  sterilized  cul- 
ture was  found  to  be  sufficient,  when  injected  intravenously, 
to  render  rabbits  susceptible  to  the  pathogenic  germ. 

In  this  connection  it  may  be  remarked  that  from  time 
to  time  statements  have  been  made  which  would  lead  us  to 
infer  that  there  are  certain  poisonous  proteids  which  in 
some  way  yet  unknown  render  the  body  especially  suscep- 
tible to  the  invasion  of  bacteria.  Rossbach  injected  a 
poisonous  albumose  from  the  juice  of  the  papain  tree  into 
the  bloodvessels  of  animals  and  obtained  a  septicaemia. 
The  blood  was  found  to  be  filled  with  non-pathogenic 
germs  which  came  from  the  intestines.  The  results  of 
RossbaCh  have,  however,  been  questioned  by  others. 
Hankin  makes  the  statement  that  a  small  dose  of  snake- 


152  BACTBEIAL    POISONS. 

poison,  which  is  too  small  to  kill  the  animal  outright,  after 
a  certain  time  may  cause  death  from  septicaemia.  He  says: 
"  The  albumose  of  the  snake-poison  has  apparently  so  far 
suppressed  the  germicidal  power  of  the  animal  that  ordinary 
decay-producing  bacteria  can  increase  and  multiply  in  the 
blood.  Further,  it  is  often  remarked  that  animals  killed 
by  a  snake-bite  putrefy  rapidly,  as  if  the  bacteria-killing 
power  of  the  blood-serum  had  been  diminished."  A  simi- 
lar statement  has  been  made  concerning  the  action  of  the 
poisonous  albumose  of  jequirity  seeds.  Further  investiga- 
tion must  discover  how  much  of  truth  and  how  little  of 
error  lie  in  these  claims. 


CHAPTER  VII. 

THE    GERMICIDAL    PROTEIDS    OF   THE    BLOOD. 

As  early  as  1872  Lewis  and  Cunningham  showed 
that  bacteria  injected  into  the  circulation  rapidly  disappear. 
In  the  blood  of  twelve  animals,  which  had  been  treated 
with  such  injections,  bacteria  could  be  found  in  only  seven 
after  six  hours.  In  thirty  animals,  bacteria  were  found  in 
the  blood  of  only  fourteen  after  twenty-four  hours,  and  in 
seventeen  animals,  bacteria  were  found  in  only  two  when 
the  examination  was  made  from  two  to  seven  days  after 
the  injection. 

In  1874,  Traube  and  Gscheidlen  found  that  the 
blood  taken  from  a  rabbit  into  the  jugular  vein  of  which 
forty-eight  hours  before  1|  c.c.  of  a  fluid  rich  in  putre- 
factive germs  was  injected,  remained  without  undergoing 
decomposition  for  months.  These  investigators  attributed 
the  germicidal  properties  of  the  blood  to  its  ozonized 
oxygen.  Similar  results  were  obtained  by  Fodor  and 
Wysokowicz.  The  latter  accounted  for  the  disappearance 
of  the  bacteria  not  by  supposing  that  they  were  destroyed 
by  the  blood,  but  that  they  found  lodgement  in  the  capil- 
laries. 

The  first  experiments  made  with  extra-vascular  blood 
were  conducted  by  Grohmann  under  the  direction  of  A. 
Schmidt.  It  was  found  that  anthrax  bacilli,  after  being 
kept  in  coagulating  plasma,  were  less  virulent,  as  shown  by. 
their  effects  upon  rabbits.  Grohmann  supposed  that  in 
some  way  the  bacteria  were  influenced  by  the  process  of 
coagulation. 

In  1887,  Fodor  made  a  second  series  of  experiments  in 
which  he  used  blood  taken  from  the  heart,  and  showed  the 
marked  germicidal  properties  of  this  on  anthrax  bacilli. 

In  1888,  Nuttall  used  defibrinated  blood  taken  from 


154  BACTERIAL    POISONS. 

various  species  of  animals  (rabbits,  mice,  pigeons,  and 
sheep)  and  found  that  this  blood  destroyed  the  bacillus 
anthracis,  bacillus  subtilis,  bacillus  megaterium,  and  staphy- 
lococcus pyogenes  aureus  when  brought  in  contact  with 
them.  Nissen  continued  this  work  and  employed  blood- 
serum  as  well  as  defibrinated  blood.  The  conclusions 
reached  were  as  follows  : 

(1)  The  addition  of  small  quantities  of  sterilized  salt- 
solution  or  bouillon  to  the  blood  does  not  destroy  its 
germicidal  properties. 

(2)  Cholera  germs  and  Eberth's  bacilli  are  easily  de- 
stroyed by  fresh  blood 

(3)  For  a  giveu  volume  of  blood  there  is  a  maximum 
amount  of  bacilli  which  can  be  added.  If  too  many 
germs  are  used  the  destruction  is  incomplete. 

(4)  Blood  whose  coagulability  has  been  destroyed  by 
the  injection  of  peptone  is  still  germicidal. 

(5)  Filtered  blood-plasma  from  the  horse  is  germicidal. 
Behrino   has  attributed   the   action   of  the  blood   of 

white  rats  on  anthrax  bacilli  to  the  presence  of  a  hypo- 
thetical basic  body  to  which  the  decidedly  alkaline  reaction 
of  the  blood  is  supposed  by  him  to  be  due.  Later,  he  lays 
special  stress  upon  the  amount  of  carbonic  acid  gas  in  the 
blood-serum. 

Buchjster  has  made  a  most  exhaustive  study  of  this 
subject,  in  which  he  has  been  aided  by  Voit,  Sittmanjst, 
and  Orthenbergee.  The  results  of  this  work  are  stated 
as  follows  : 

(1)  The  germicidal  action  of  the  blood  is  not  due  to 
phagocytes,  because  it  is  not  influenced  by  freezing  and 
thawing  the  blood,  by  which  the  leucocytes  of  the  blood 
of  the  rabbit  are  destroyed. 

(2)  The  germicidal  properties  of  the  cell-free  serum  must 
be  due  to  soluble  constituents. 

(3)  Neither  neutralization  of  the  serum,  nor  the  addition 
of  pepsin,  nor  the  removal  of  carbonic  acid,  nor  treatment 
with  oxygen  have  any  effect  upon  the  germicidal  proper- 
ties of  the  blood. 

(4)  Dialysis  of  the  serum  against  water   destroys   its 


GERMICIDAL    PROTEIDS    OF    THE    BLOOD.      155 

activity,  while  dialysis  against  0.75  per  cent,  salt  solution 
does  not.  In  the  diffusate  there  is  no  germicidal  substance. 
The  loss  by  dialysis  with  water  must  be  due  to  the  with- 
drawal of  the  inorganic  salts  of  the  serum. 

(5)  The  same  is  shown  to  be  the  case  when  the  serum  is 
diluted  with  water  and  when  it  is  diluted  with  the  salt 
solution.  In  the  former  the  germicidal  action  is  destroyed, 
while  in  the  latter  it  is  not. 

(6)  The  inorganic  salts  have  in  and  of  themselves  no 
germicidal  action.  They  are  active  only  in  so  far  as  they 
affect  the  normal  properties  of  the  albuminates  of  the  serum. 
The  germicidal  properties  of  the  serum  reside  in  its  proteid 
constituents. 

(7)  The  difference  in  the  effects  of  the  active  serum  and 
that  which  has  been  heated  to  55°  is  due  to  the  altered 
condition  of  the  albuminates.  This  difference  may  possibly 
be  a  chemical  one  (due  to  changes  within  the  molecule),  or  it 
may  be  due  to  alterations  in  mycelial  construction.  The 
albuminates  act  on  the  bacteria  only  when  the  former  are 
in  an  "active  state." 

Halliburton  has  prepared  from  the  lymphatic  glands 
a  globulin  which  he  designates  as  cell-globulin  /?,  and  which 
agrees  with  fibrin  ferment  in  inducing  coagulation  in  plasma. 
Hankin  has  tested  the  germicidal  properties  of  this  cell- 
globulin.  His  experiments  have  been  conducted  in  the 
following  manner  :  The  lymphatic  glands  (in  later  experi- 
ments the  spleen,  also)  of  a  dog  or  cat  are  freed  as  much 
as  possible  from  fat  and  connective  tissue,  then  finely 
divided,  and  extracted  with  a  dilute  solution  of  sodium 
sulphate  (one  part  of  a  saturated  sodium  sulphate  solution 
+  nine  parts  of  water).  The  cell-globulin  passes  into 
solution,  while  the  other  proteids  are  but  sparingly  soluble. 
After  twenty-four  hours  the  fluid  is  filtered  and  mixed 
with  an  excess  of  alcohol.  The  voluminous  precipitate 
containing  the  cell-globulin  is  collected  on  a  filter  and 
washed  with  absolute  alcohol.  For  use,  a  part  is  dissolved 
in  water  and  a  small  quantity  of  a  bouillon  culture  of  the 
anthrax  bacillus  added.  Plate  cultures  are  made  along 
with  control  plates  from  time  to  time,  and  in  this  way  the 


156  BACTERIAL    POISONS. 

germicidal  property  of  the  substance  is  demonstrated. 
Hankin  closes  this  contribution  with  the  following  con- 
clusions : 

(1)  Halliburton's  cell-globulin  /5  has  marked  germi- 
cidal properties. 

(2)  In  this  respect  it  differs  from  fibrin  ferment. 

(3)  The  germicidal  property  of  this  substance  seems  to 
be  identical  with  that  of  serum  as  described  by  Buchner, 
Nissen,  and  Nuttall. 

(4)  The  active  properties  of  the  serum  are  probably  due 
to  this  or  to  an  allied  body. 

In  a  more  recent  contributiou  Hankin  designates  the 
germicidal  agents  of  the  body  as  "defensive  proteids." 
He  thinks  it  probable  that  blood-serum  owes  its  activity 
to  these  bodies  and  that  the  assumption  of  an  "active  con- 
dition of  the  serum  albuminate"  made  by  Buchner  is 
unnecessary.  He  also  thinks  that  Behring's  supposed 
alkaline  base  exists  in  the  form  of  an  albumose.  We  know 
of  three  albumoses  which  are  alkaline  in  reaction.  These 
are  the  protomyosinose  and  deuteromyosinose  of  Kuhne 
and  Chittenden,  prepared  by  the  digestion  of  myosin, 
and  the  anthrax  albumose  of  Martin. 

By  a  method  similar  to  that  which  he  had  employed  in 
the  preceding  experiments,  Hankin  has  isolated  a  "defen- 
sive proteid  "  from  the  blood-serum  and  the  spleen  of  the 
rat.  This  substance  belongs  to  the  globulins,  and  the  nat- 
ural immunity  of  the  rat  against  anthrax  is  probably  due 
to  its  existence  in  the  blood. 

Stern  finds  that  the  blood  taken  from  different  men,  or 
from  the  same  man  at  different  times,  varies  markedly  in 
its  germicidal  properties ;  also,  that  the  germicidal  proper- 
ties of  the  blood  when  kept  at  42°  are  at  least  as  great  as 
at  the  normal  temperature  of  the  body.  These  statements 
are  substantially  confirmed  by  Rovighi. 


CHAPTER  VIII. 

METHODS    OF    EXTRACTING    PTOMAINES. 

From  what  has  been  given  in  the  preceding  pages,  one 
may  gather  some  idea  of  the  peculiar  difficulties  with  which 
the  chemist  has  to  contend  in  his  endeavors  to  isolate  the 
basic  products  of  putrefaction.  He  has  to  deal  with  very 
complex  substances,  of  the  nature  and  reactions  of  many 
of  which  he  must  be  ignorant.  Besides,  the  substances 
which  he  seeks  are  often  most  prone  to  undergo  decompo- 
sition, and  in  this  way  escape  detection.  Many  ptomaines 
are  volatile  or  decomposable  at  any  temperature  near  that 
of  boiling  water.  In  these  cases,  solutions  cannot  be 
evaporated  in  the  ordinary  way  and  the  poison  separated 
from  the  residue.  Indeed,  the  investigator  has  frequently 
been  disappointed  when  on  the  evaporation  of  a  solution, 
which  he  has  demonstrated  to  be  poisonous,  he  finds  that 
the  residue  is  wholly  inert.  Again,  he  may  destroy  the 
ptomaine  by  the  action  of  reagents  which  he  uses.  So 
simple  a  procedure  as  the  removal  of  a  metallic  base  from 
a  solution  containing  a  ptomaine,  by  precipitation  with 
hydrogen  sulphide  gas,  has  been  known  to  destroy  wholly 
the  ptomaine.  Probably  the  most  perplexing  difficulty  in 
the  isolation  of  these  putrefactive  alkaloids  lies  in  the  great 
number,  complexity,  and  diversity  of  the  other  substances 
present  in  the  decomposing  mass.  The  same  ptomaine  may 
be  present  in  equal  quantities  in  two  samples  of  milk,  and 
yet  it  may  be  easily  obtained  from  the  one,  while  from  the 
other  only  minute  traces  can  be  secured.  The  difference  is 
due  to  the  fact  that  the  other  constituents  of  the  milk  in 
the  two  samples  are  at  different  stages  of  the  putrefactive 
process,  and,  consequently,  differ  greatly  in  their  reactions 
and  in  their  effects  upon  the  agents  employed  to  isolate 
the  poison.     All  chemists  will  appreciate  these  difficulties. 


158  BACTERIAL    POISONS. 

One  of  the  first  things  for  the  chemist  who  undertakes 
to  do  this  work  is  to  ascertain  whether  or  not  his  reagents 
are  pure.  We  have  found  a  number  of  samples  of  German 
ether,  which  was  imported  on  account  of  its  supposed 
purity,  to  yield  on  spontaneous  evaporation  a  residue  which 
gave  several  of  the  alkaloidal  reactions,  and  a  few  drops  of 
which,  injected  under  the  skin  of  a  frog,  caused  paralysis 
and  death  within  a  few  minutes.  We  would  advise  that  500 
c.c.  of  the  ether  to  be  used  should  be  allowed  to  evaporate 
spontaneously,  and  its  residue,  if  there  be  one,  be  examined 
both  chemically  and  physiologically.  The  basic  substance 
which  exists  in  some  samples  of  sulphuric  ether  is  pyridine. 

Guareschi  and  Mosso  found  commercial  alcohol  almost 
invariably  to  contain  small  quantities  of  an  alkaloidal  sub- 
stance, the  odor  of  which  is  similar  to  that  of  nicotine  and 
pyridine.  Its  solutions  are  precipitated  by  gold  chloride, 
phosphowolframic  acid,  phosphomolybdic  acid,  potassium 
iodide,  and  Mayer's  reagent,  but  not  by  platinum  chloride 
or  tannic  acid.  It  does  not  reduce,  or  reduces  feebly,  ferric 
salts.  From  one  sample  of  alcohol  they  obtained  a  base 
which,  in  addition  to  the  above  reactions,  did  give  a  pre- 
cipitate with  platinum  chloride.  Alcohol  may  be  freed 
from  these  substances  by  distillation  over  tartaric  acid. 

In  amylic  alcohol,  Haitinger  has  found  as  much  as 
0.5  per  cent,  of  pyridine.  It  may  be  purified  in  the  same 
manner  as  recommended  for  ethylic  alcohol. 

Chloroform,  when  found  to  leave  any  residue  on  evapora- 
tion, should  be  washed  first  with  distilled  water,  then  with 
distilled  water  rendered  alkaline  with  potassium  carbonate, 
then  dried  over  calcium  chloride  and  distilled. 

Petroleum  ether  sometimes  contains  a  base  which  has  an 
odor  similar  to  trimethylamine  or  pyridine,  and  which  gives 
a  precipitate  with  platinum  chloride,  forming  in  octahedra. 

Benzole  may  contain  a  similar  substance. 

The  following  methods  have  been  used  for  the  purpose 
of  extracting  the  putrefactive  alkaloids  : 

The  Stas-Otto  Method. — This  method  depends  upon 
the  following  facts  :  (1)  The  salts  of  the  alkaloids  are  sol- 


METHODS    OF    EXTRACTING    PTOMAINES.        159 

uble  in  water  and  alcohol,  and  generally  insoluble  in  ether, 
and  (2)  the  free  alkaloids  are  soluble  in  ether,  and  are  re- 
moved from  alkaline  fluids  by  agitation  with  ether.  These 
principles  are  capable  of  great  variety  in  their  application. 
The  usual  directions  are  as  follows  :  Treat  the  mass  under 
examination  with  about  twice  its  weight  of  pure  90  per 
cent,  alcohol,  and  from  ten  to  thirty  graius  of  tartaric  or 
oxalic  acid;  digest  the  whole  for  some  time  at  about  70°, 
and  filter.  Evaporate  the  filtrate  at  a  temperature  not  ex- 
ceeding 35°  either  in  a  strong  current  of  air  or  in  vacuo 
over  sulphuric  acid.  Take  up  the  residue  with  absolute 
alcohol,  filter,  and  again  evaporate  at  a  low  temperature. 
Dissolve  this  residue  in  water,  render  alkaline  with  sodium 
bicarbonate,  and  agitate  with  ether.  After  separation  re- 
move the  ether  with  a  pipette,  or  by  means  of  a  separator, 
and  allow  it  to  evaporate  spontaneously.  The  residue  may 
be  further  purified  by  redissolving  in  water  and  again  ex- 
tracting with  ether. 

The  following  modifications  of  this  method  are  em- 
ployed :  Instead  of  tartaric  or  oxalic  acid,  acetic  acid  is 
frequently  used. 

When  the  fluid  suspected  of  containing  a  ptomaine  is 
already  acid  from  the  development  of  lactic  or  other  organic 
acid,  the  addition  of  an  acid  is  often  dispensed  with. 

Ether  extracts  are  made  from  both  acid  and  alkaline 
solutions. 

Chloroform,  amylic  alcohol,  and  benzine  are  used  as  sol- 
vents after  extraction  with  ether. 

The  modification  of  this  method,  as  carried  out  by  Selmi 
and  Marino-Zuco  is  given  in  detail  as  follows : 

The  material  is  divided  as  minutely  as  possible,  placed 
in  a  large  flask,  and  treated  with  twice  its  volume  of  90  per 
cent,  alcohol,  and  acidulated  with  tartaric  acid  in  the  pro- 
portion of  0.5  gramme  to  100  c.c.  of  the  mixture,  taking 
care  from  time  to  time  that  the  reaction  is  permanently 
acid.  The  flask,  which  is  connected  with  a  reflux  condenser, 
is  now  placed  on  the  water-bath  and  kept  at  the  constant 
temperature  of  70°  for  twenty-four  hours.  While  yet 
warm  the  liquid  is  transferred  to  a  special  apparatus  for 


160  BACTERIAL    POISOXS. 

filtration  by  the  aid  of  atmospheric  pressure.  The  liquid 
is  poured  upon  a  wet  cloth  supported  upon  a  perforated 
porcelain  funnel,  which  is  connected  below  with  a  receiver 
exhausted  by  a  water-pump  or  aspirator.  In  this  way 
rapid  filtration  is  secured,  and  by  repeated  washing  the 
extraction  is  made  thorough.  The  acid  alcoholic  liquid  is 
now  transferred  to  a  special  distillation  apparatus. 

A  large  tubulated  retort  of  ten  litres  capacity  is  con- 
nected by  means  of  a  cork  to  a  large  tubulated  receiver. 
The  tubulure  of  the  retort  is  provided  with  a  small  per- 
forated cork,  which  carries  a  glass  tube  finely  drawn  out 
and  extending  to  the  bottom  of  the  retort.  The  tubulure 
of  the  receiver  is  connected  with  Liebig's  bulbs  containing 
dilute  sulphuric  acid  (1  to  10),  and  the  bulbs  in  turn  are 
connected  with  a  water-pump  or  aspirator. 

In  order  to  prevent  the  passage  of  air  through  the  corks, 
they  are  covered  with  animal  membrane  which  has  been 
freed  from  fat.  By  means  of  the  aspirator  a  fine  current 
of  air  is  drawn  through  the  liquid  and  suffices  to  keep  it 
constantly  agitated.  The  retort  is  kept  on  the  water-bath 
at  a  temperature  of  from  28°  to  30°.  The  receiver  is  kept 
cold  by  a  current  of  water  In  this  manner  the  distilla- 
tion of  the  alcohol  goes  on  rapidly  and  conveniently.  More- 
over, decomposition  is  so  far  prevented  that  volatile  bases 
are  never  found  in  the  bulbs. 

The  aqueous  residue,  after  the  removal  of  the  alcohol  by 
distillation,  is  filtered  and  extracted  with  ether  as  long  as 
anything  is  dissolved.  It  is  then  mixed  with  powdered 
glass  and  evaporated  to  dryness  in  vacuo.  This  residue  is 
repeatedly  extracted  with  absolute  alcohol.  The  alcohol  is 
distilled  again  in  the  apparatus  already  described.  The 
residue  is  taken  up  with  distilled  water  and  filtered.  It  is 
then  made  alkaline  with  sodium  bicarbonate  and  repeatedly 
extracted  with  ether,  benzine,  and  chloroform. 

In  order  to  obtain  the  base  from  the  solvent,  the  greater 
part  may  be  evaporated  on  the  water-bath  and  the  re- 
maiuder  allowed  to  evaporate  spontaneously,  or  the  re- 
mainder may  be  treated  with  dilute  hydrochloric  acid  and 
the  evaporation  continued  on  the  water-bath  or  in  vacuo. 


beieger's  method.  161 

Dragendorff's  Method. — The  finely  divided  sub- 
stance is  digested  for  some  hours  with  water  acidulated 
with  sulphuric  acid  at  from  40°  to  50°.  This  is  repeated 
two  or  three  times,  and  the  united  filtered  extracts  are 
evaporated  to  a  syrup.  This  is  treated  with  four  volumes 
of  alcohol  and  digested  for  twenty- four  hours  at  30.°  After 
cooling,  the  alcoholic  extract  is  filtered,  the  residue  washed 
with  70  per  cent,  alcohol,  aud  the  united  filtrates  freed 
from  alcohol  by  distillation.  The  aqueous  residue,  diluted 
if  desirable,  is  filtered  and  submitted  to  the  following  ex- 
tractions : 

(1)  The  acid  liquid  is  shaken  with  freshly  rectified  petro- 
leum ether  as  long  as  this  reagent  leaves  any  residue  on 
evaporation. 

(2)  The  acid  fluid  is  now  extracted  with  benzine. 

(3)  The  next  solvent  used  is  chloroform. 

(4)  The  liquid  is  now  again  extracted  with  petroleum 
ether  in  order  to  remove  traces  of  benzine  and  chloroform. 

(5)  The  liquid  is  now  made  alkaline  with  ammonia  and 
successively  extracted  with  petroleum  ether,  benzine,  chloro- 
form, aud  amylic  alcohol. 

(6)  The  remainder  of  the  ammoniacal  liquid  is  mixed 
with  powdered  glass,  evaporated  to  dryness,  the  residue 
pulverized,  and  extracted  with  chloroform. 

The  residue  obtained  with  each  of  the  above  solvents 
should  be  examined  for  ptomaines. 

Brieger's  Method. — The  substance  under  examination 
is  divided  as  finely  as  possible,  aud  then  heated  with  water 
slightly  acidified  with  hydrochloric  acid.  During  the 
heating  care  must  be  taken  that  the  feebly  acid  reaction  is 
maintained.  The  heating  should  continue  for  only  a  few 
minutes.  The  liquid  is  then  filtered  aud  concentrated,  at 
first  on  a  plate  and  then  on  the  water-bath,  to  a  syrup.  If 
one  has  material  which  is  highly  odorous,  as  is  the  case 
frequently  both  with  aqueous  and  alcoholic  extracts  of 
putrid  material,  Brieger  recommends  that  a  piece  of 
apparatus  devised  by  Bocklisch  be  used.  The  fluid  to  be 
evaporated  is  placed  in  a  globular  flask,  the  rubber  stopper 


162 


BACTERIAL    POISONS, 


of  which  carries  two  small  glass  tubes.  One  of  these,  b, 
extends  to  the  bottom  of  the  flask,  while  A  terminates  just 
above  the  surface  of  the  liquid.  The  tube,  A,  is  connected 
with  a  water-pump  or  aspirator,  which  draws  the  vapor 
through  the  tube.  In  order  to  prevent  the  return  of  con- 
densed fluids,  the  end  of  A  in  the  flask  is  curved  upon 
itself.  The  tube,  b,  is  finely  drawn  out  and  through  it  a 
current  of  air  is  constantly  moving.  This  prevents  the 
formation  of  a  deposit  or  a  pellicle  in  the  fluid.     By  regu- 


lating the  amount  of  air  coming  through  this  tube,  more 
or  less  of  a  vacuum  will  be  formed  in  the  flask.  After 
evaporation  to  a  syrup,  au  extraction  is  made  with  96  per 
cent,  alcohol,  and  the  filtered  extract  is  treated  with  a  warm 
alcoholic  solution  of  lead  acetate.  The  lead  precipitate  is 
removed  by  filtration,  the  filtrate  evaporated  to  a  syrup  and 
again  extracted  with  96  per  cent,  alcohol.  The  alcohol  is 
driven  off ;  the  residue  taken  up  with  water;  traces  of 
lead  removed  with  hydrogen  sulphide ;  and  the  filtrate, 
acidified  with  hydrochloric  acid,  evaporated  to  a  syrup. 
This  syrup  is  extracted  with  alcohol,  and  the  filtrate  pre- 


METHODS    OF    GAUTIER    AXD    ETARD.  163 

cipitated  with  an  alcoholic  solution  of  mercuric  chloride. 
The  mercury  precipitate  is  boiled  with  water,  and  on  ac- 
count of  differences  in  solubility  of  the  double  compounds 
with  mercury,  one  ptomaine  may  be  separated  from  others 
at  this  stage  of  the  process.  (If  thought  best,  the  lead  pre- 
cipitate may  be  freed  from  lead  aud  carried  through  the 
following  steps  of  the  process.  Brieger  has  found  small 
amounts  of  ptomaines  in  the  lead  precipitate  only  in  his 
work  with  poisonous  mussels.) 

The  mercury  nitrate  is  freed  from  mercury,  evaporated, 
aud  the  excess  of  hydrochloric  acid  carefully  neutralized 
with  soda  (the  reaction  is  kept  feebly  acid),  then  it  is  again 
taken  up  with  alcohol  in  order  to  free  it  from  inorganic 
salts.  The  alcohol  is  evaporated,  the  residue  taken  up  with 
water,  the  remaining  traces  of  hydrochloric  acid  neutralized 
with  soda ;  the  whole  acidified  with  nitric  acid,  and 
treated  with  phosphomolybdic  acid.  The  phosphomolyb- 
date  double  compound  is  separated  by  nitration,  and  de- 
composed by  neutral  acetate  of  lead.  This  is  hastened 
by  heating  on  the  water-bath.  The  lead  is  removed  by 
hydrogen  sulphide,  the  filtrate  is  evaporated  to  a  syrup  and 
taken  up  with  alcohol,  from  which  many  ptomaines  are 
deposited  as  chlorides,  or  double  salts  may  be  formed  in 
the  alcoholic  solution.  Brieger  states  that  the  chlorides 
as  deposited  from  the  alcoholic  solution  are  seldom  pure, 
and  he  advises  for  their  purification,  precipitation  with 
gold  chloride,  platinum  chloride,  or  picric  acid,  and,  on 
account  of  differences  in  solubility  of  these  double  salts, 
the  process  of  purification  is  rendered  more  easy.  The 
chloride  of  the  base  is  obtained  by  removing  the  metallic 
base  with  hydrogen  sulphide;  while  the  picrate  is  taken 
up  with  water,  acidified  with  hydrochloric  acid,  and  re- 
peatedly extracted  with  ether,  in  order  to  remove  the 
picric  acid. 

The  Methods  of  Gautier  and  Etard. — The  putrid 
matters,  liquid  and  solid,  are  distilled  at  a  low  temperature 
in  vacuo.  The  distillate  (A)  contains  a  considerable  quan- 
tity of  ammonium  carbonate,  some  phenol,  skatol,  trimethyl- 


164  BACTERIAL    POISONS. 

amine,  and  the  volatile  fatty  acids.     The  residue  after  dis- 
tillation is  treated  in  succession  by  ether  and  by  alcohol. 

The  extraction  with  ether  (B)  separates  the  ptomaines 
and  some  fatty  acids.  The  alcoholic  extract  (C)  removes 
the  remainder  of  the  fatty  acids,  as  well  as  the  acid  and 
neutral  nitrogenized  bodies,  almost  all  of  which  are  crys- 
tallizable.  The  insoluble  residue  is  boiled  with  dilute 
hydrochloric  acid,  with  exclusion  of  air,  finally  evaporated 
to  dryness,  and  the  residue  again  extracted  with  alcohol. 
This  new  alcoholic  solution  (D)  can  be  divided  by  acetate 
and  subacetate  of  lead  into  two  principal  portions. 

By  operating  in  this  manner  the  complex  products  of 
putrefaction  are  readily  separated  into  four  portions. 

In  his  more  recent  work,  Gautier  has  employed  the 
following  method  :  The  putrid  liquids,  after  the  removal 
of  fats,  are  feebly  acidified  with  very  dilute  sulphuric  acid, 
then  distilled  in  vacuo  at  a  low  temperature.  The  distillate 
contains  ammonia,  phenol,  iudol,  and  skatol.  The  syrupy 
residue,  separated  from  any  crystals  which  may  have 
formed,  is  rendered  alkaline  with  baryta,  filtered,  and  ex- 
tracted a  great  number  of  times  with  chloroform,  in  order 
to  dissolve  the  bases.  The  solution  is  distilled  at  a  low 
temperature,  either  in  vacuo  or  in  a  current  of  carbonic 
acid.  The  contents  of  the  retort,  on  being  treated  with 
water  and  tartaric  acid,  separate  into  a  brown  resin  and  a 
liquid  portion.  The  latter  is  removed  and  treated  with  a 
dilute  solution  of  potash,  when  it  gives  oif  the  odor  of 
carbylamine,  which  was  discovered  by  Gautier  in  1866, 
and  which,  according  to  Calmel,  is  a  constituent  of  the 
venom  of  toads.  The  alkali  also  sets  free  the  bases,  which 
are  removed  by  extraction  with  ether,  and  the  ether  evapo- 
rated in  a  current  of  carbonic  acid  gas  under  slight  pressure, 
theu  under  a  bell-jar  over  caustic  potash.  The  bases  may 
be  separated  by  fractional  precipitation  with  platinum 
chloride,  or,  if  present  in  sufficient  quantity,  by  distillation 
in  vacuo. 

Still  later,  Gautier  has  modified  his  method  as  follows : 
The  alkaline  putrid  liquid  is  treated  with  oxalic  acid  (in- 
stead of  sulphuric  acid)  to  free  acidulation  and  as  long  as 


REMARKS    UPON    THE    METHODS.  165 

the  fatty  acids  continue  to  separate.  The  liquid  is  then 
warmed  and  distilled  as  long  as  a  turbid  fluid  passes  over. 
Pyrrol,  skatol,  phenol,  indol,  volatile  fatty  acids,  and  some 
of  the  ammonia  pass  over.  The  portion  which  remains  in 
the  retort  is  rendered  alkaline  with  lime-water.  The  pre- 
cipitate which  forms,  and  which  contains  the  greater  part 
of  the  fixed  fatty  acids,  is  removed.  The  liquid  portion, 
which  is  alkaline,  is  distilled  to  dryness,  care  being  taken 
to  receive  the  distillate  in  very  dilute  sulphuric  acid.  The 
bases  aud  ammonia  pass  over.  The  distillate  is  neutralized 
(with  sulphuric  acid)  and  evaporated  almost  to  dryness, 
then  decanted  from  ammonium  sulphate,  which  crystallizes. 
The  mother-liquor  is  extracted  with  concentrated  alcohol, 
which  dissolves  the  sulphates  of  the  ptomaines.  After 
driving  off  the  alcohol,  the  residue  is  rendered  alkaline 
with  caustic  soda,  and  successively  extracted  with  ether, 
petroleum  ether,  and  chloroform. 

The  lime  precipitate  is  dried  and  extracted  with  ether 
of  thirty-six  degrees,  which  removes  any  fixed  bases  that 
may  be  present. 

Remarks  upon  the  Methods.  —  The  fundamental 
difference  between  the  Stas-Otto  and  the  Dragendorff 
methods  consists  in  the  fact  that  in  the  former  the  first 
extraction  is  made  with  a  dilute  solution  of  an  organic  acid 
(tartaric  usually),  while  in  the  second  a  similar  solution  of 
a  mineral  acid  (sulphuric)  is  employed.  In  their  various 
modified  forms  any  solvent  may  be  used  for  separating  the 
alkaloid  from  the  other  constituents  of  the  original  solu- 
tion. Therefore,  the  question  has  been  asked,  Which  is 
the  more  suitable  acid  for  use  in  making  the  first  solution? 
The  answer  to  this  question  will  also  be  the  one  to  the 
question,  Which  is  the  better  method  of  extracting  pto- 
maines, the  Stas-Otto  method  or  that  of  Dragendorff? 
The  Italian  chemists  Guareschi  and  Mosso  have  at- 
tempted to  answer  this  question  experimentally,  and  the 
evidence  which  they  have  furnished  is  condemnatory  of 
the  method  of  Dragendorff.  They  show  that  basic 
bodies  are  formed  by  the  action  of  the  dilute  sulphuric 

8* 


166  BACTERIAL    POISONS. 

acid  upon  albuminous  substances.  As  this  poiut  is  of 
vital  importance  to  the  investigator  in  this  branch  of 
chemical  science,  we  will  give  a  brief  abstract  of  the  work 
of  Guareschi  and  Mosso  : 

One  kilogramme  of  fresh  meat  was  treated  with  dilute  sul- 
phuric acid  (in  the  proportion  recommended  in  the  Dra- 
gendorff  method)  and  alcohol.  The  dark  solution  after 
filtration  was  made  alkaline  with  ammonium  hydrate  and 
extracted  with  ether.  The  ethereal  solution  gave  on  evap- 
oration an  oily  substance  which  had  the  odor  of  extracts 
obtained  from  putrid  fibrin.  This  substance,  which  was 
obtained  in  considerable  quantity,  was  soluble  in  water  and 
strongly  alkaline  in  reaction.  After  neutralization  with 
hydrochloric  acid,  its  aqueous  solutions  gave  the  following 
alkaloidal  tests : 

(1)  With  platinum  chloride,  a  yellowish-red  precipitate, 
insoluble  in  water,  alcohol,  and  ether,  and  apparently  iden- 
tical with  the  compound  obtained  from  putrid  fibrin  with 
the  same  reagent. 

(2)  With  gold  chloride,  yellow  precipitate,  then  reduc- 
tion to  metallic  gold. 

(3)  With  phosphomolybdic  acid,  a  heavy,  yellow  precipi- 
tate, forming  a  blue  solution  on  the  addition  of  ammonium 
hydrate. 

(4)  With  phosphotungstic  acid,  a  white  precipitate. 

(5)  Writh  Mayer's  reagent,  a  heavy,  whitish  precipitate. 

(6)  With  picric  acid,  white  precipitate,  instantly. 

(7)  With  iodine  in  potassium  iodide  solution,  a  heavy 
kermes-red  precipitate. 

(8)  With  tannic  acid,  white  precipitate. 

(9)  With  mercuric  chloride,  white,  amorphous  precipi- 
tate. 

(10)  With  Marme's  reagent,  heavy  precipitate. 

(11)  With  potassium  ferricyanide,  no  precipitate,  but  a 
cloudiness,  with  a  formation  of  Prussian  blue  on  the  addi- 
tion of  ferric  chloride. 

The  same  quantity  of  this  meat  was  also  treated  by  the 
Stas-Otto  method.  The  alcoholic  extract  was  evaporated 
on  the  water- bath  and  not  in  vacuo.     The  acid  was  neu- 


EEMARKS    UPON    THE    METHODS.  167 

tralized  with  sodium  bicarbonate.  The  ether  extract  gave 
on  evaporation  a  faintly  yellow  residue,  of  not  unpleasant 
odor  and  feebly  alkaline  reaction.  After  neutralization 
with  hydrochloric  acid,  it  was  only  slightly  soluble  in 
water.  The  pale  yellow  filtrate  gave  no  precipitate  with 
Nos.  1,  2,  8,  9,  and  10  of  the  above-mentioned  reagents, 
but  gave  a  slight  turbidity  with  Nos.  3,  4,  5,  6,  and  7,  and 
with  11  formed  Prussian  blue. 

Guareschi  and  Mosso  conclude  from  this  and  other 
experiments  that  the  Dragendorff  method  is  not  suit- 
able for  the  extraction  of  ptomaines,  and  they  recommend 
the  employment  of  the  Stas-Otto  method  with  these  con- 
ditions :  (1)  no  more  acid  should  be  added  than  is  abso- 
lutely necessary  to  keep  the  reaction  acid  ;  (2)  the  heat 
used  in  evaporation  should  not  be  great,  and  it  is  better 
that  evaporation  should  be  made  in  vacuo.  In  this  way, 
they  say,  no  ptomaine  will  be  obtained  from  fresh  tissue. 

The  same  investigators  extracted  fresh  flesh  without  the 
addition  of  any  acid.  Thirty  kilogrammes  of  perfectly  fresh 
meat  were  digested  for  two  hours  at  from  50°  to  60°  with 
about  one  and  one-half  volumes  of  water.  The  fluids  of  the 
meat  contained  enough  acid  to  give  to  the  whole  of  this  solu- 
tion an  acid  reaction.  It  was  evaporated  to  half  its  volume 
on  the  water-bath,  filtered,  and  evaporated  still  further. 
The  small  residue  was  taken  up  with  about  four  volumes 
of  96  per  cent,  alcohol.  The  reddish,  alcoholic  solution 
left  on  evaporation  on  the  water-bath  a  brownish  residue, 
which  was  dissolved  in  water  and  extracted  with  ether  (A), 
then  the  solution  was  made  alkaline  with  ammonium 
hydrate  and  again  extracted  with  ether  (B). 

A  gave  on  evaporation  and  cooling  crystals  of  methyl- 
hydantoin,  while  the  mother-liquor  contained  acetic  acid. 

B  also  yielded  crystals  of  methyl-hydantoin,  while  the 
mother-liquor  gave  alkaloidal  reactions  with  most  of  the 
general  alkaloidal  reagents,  none  with  platinum  chloride. 
Methyl-hydantoin  does  not  give  these  reactions. 

Marino- Zuco  has  made  many  comparative  tests  with 
these  two  methods.  He  ascertained  that  by  treating  fresh 
eggs,  brain,  liver,  spleen,  kidney,  lungs,  heart,  and  blood 


168  BACTERIAL    POISONS. 

by  either  of  the  methods,  he  could  obtain  a  substance  which 
gave  alkaloidal  reactions,  and  which  he  demonstrated  to  be 
choline.  His  experiments  led  him  to  believe  that  choline 
did  not  exist  pre-formed  in  these  fresh  tissues,  but  that  it 
resulted  from  the  action  of  the  dilute  acids  upon  lecithin. 
It  was  found  most  abundantly  in  those  tissues  which  are 
rich  in  lecithin,  such  as  the  yolks  of  eggs,  brain,  liver, 
and  blood ;  while  only  traces  could  be  obtained  from  the 
whites  of  eggs,  lungs,  and  heart.  The  method  of  Dragen- 
dorff  was  found  to  furnish  much  larger  quantities  of 
choline  than  could  be  obtained  by  the  StaS-Otto  method. 

Coppola  agrees  with  his  countrymen,  mentioned  above, 
in  condemning  the  method  of  Dragendorff. 

Enough  has  been  said  to  show  that  results  obtained  by 
the  Stas-Otto  method  are  much  more  reliable  than  those 
secured  by  the  method  of  Dragendorff.  However,  the 
former  is  not  a  perfect  method,  nor  has  a  perfect  one  yet 
been  devised.  The  principal  difficulties  met  with  in  the 
Stas-Otto  method  are  as  follows  : 

(1)  In  most  instances  the  extraction  of  the  base  is  very 
incomplete.  (2)  The  degree  to  which  the  putrefactive 
alkaloid  is  removed  by  the  solvent  will  depend  very 
largely  upon  the  nature  of  the  other  substances  present. 
This  fact  in  some  cases  aids  and  in  others  hinders  the 
labors  of  the  investigator.  Thus,  several  ptomaines,  which 
when  pure  are  wholly  insoluble  in  ether,  may  be  removed, 
in  part  at  least,  from  organic  mixtures  by  this  solvent  by 
passing  into  the  solution  along  with  other  substances,  but 
if  the  attempt  is  made  to  purify  one  of  these  bases  by  re- 
peated solution  and  extraction  with  ether,  the  result  is  a 
failure,  because  the  more  perfectly  the  alkaloid  is  freed 
from  impurities,  the  less  soluble  it  is  in  ether.  This  criti- 
cism, however,  is  equally  applicable  to  the  Dragendorff 
method,  and  to  all  others  in  so  far  as  extractions  are  made. 

However,  we  may  state  that  whenever  it  is  applicable 
this  method  is  the  best  now  employed.  By  it  the  sub- 
stances are  submitted  to  the  least  chemical  manipulation, 
and  the  results  obtained  are  the  most  reliable.  Many  of 
the  more  complex  putrefactive  products  are  so  easily  de- 


REMARKS    UPON    THE    METHODS  169 

composed  or  otherwise  altered  that  the  investigator  should 
seek  to  isolate  them  by  the  simplest  methods  possible.  If 
it  can  be  done  without  the  addition  of  any  acid  or  without 
the  application  of  heat,  so  much  the  better. 

Especially  is  the  modification  of  this  method  employed 
by  Marino-Zuco,  and  already  described,  to  be  commended. 

By  his  method,  Brieger  has  discovered  a  considerable 
number  of  basic  bodies  and  has  given  great  impetus  to  the 
study  of  the  chemistry  of  putrefaction.  The  method  is 
capable  of  a  great  many  modifications.  As  long  ago  as  1868, 
Bergmans  and  Schmiedeberg  employed  precipitation 
with  metallic  salts  in  order  to  obtain  sepsine  from  putrid 
yeast.  The  method  used  by  them  was  as  follows  :  Putrid 
yeast  was  diffused  through  parchment  paper ;  the  diffusate 
was  acidified  with  hydrochloric  acid,  and  treated  with  mer- 
curic chloride  solution  until  a  heavy  cloudiness  and,  after 
some  time,  a  slight  precipitate  formed.  This  was  removed 
by  filtration ;  the  filtrate  was  rendered  strongly  alkaline 
with  sodium  carbonate,  and  then  further  treated  with  a 
solution  of  mercuric  chloride  as  long  as  a  precipitate 
formed.  This  precipitate  was  collected  on  a  filter,  washed, 
suspended  in  a  little  acidified  water,  and  decomposed  with 
hydrogen  sulphide.  The  precipitate  was  removed,  the  free 
hydrochloric  acid  in  the  filtrate  taken  up  with  silver  car- 
bonate, and  the  excess  of  silver  removed  with  hydrogen 
sulphide.  The  filtrate  was  evaporated  to  dryness";  the 
residue  dissolved  in  alcohol  (a  part  remaining  insoluble), 
and  acidified  with  sulphuric  acid,  when  a  colorless  or 
slightly  yellow  crystalline  precipitate  formed.  The  crys- 
talline sepsine  sulphate  was  purified  by  solution  in  water 
and  precipitation  with  alcohol. 

Brieger  has  obtained  some  of  his  bases  by  a  much  sim- 
plified modification  of  his  complete  method,  which  we  have 
given  in  full.  For  instance,  in  obtaining  neuridine,  he 
treated  the  aqueous  extract  of  the  putrid  material,  after 
boiling  and  filtration,  with  mercuric  chloride,  collected  the 
precipitate,  decomposed  it  with  hydrogen  sulphide,  evapor- 
ated the  filtrate  on  the  water-bath,  and  extracted  the  base 
from  the  residue  with  dilute  alcohol. 


170  BACTERIAL    POISONS. 

By  this  method  and  its  modifications  Brieger  has 
obtained  many  brilliant  results,  among  which  maybe  men- 
tioned his  discovery  of  mytilotoxine,  typhotoxine,  and 
tetauine.  However,  the  method  is  not  free  from  criticism. 
The  great  number  of  chemical  manipulations  to  which  the 
organic  matter  is  subjected  is  liable  to  lead  to  the  formation 
of  some  basic  substances  and  to  the  destruction  of  others. 
One  is  justified  in  considering  the  isolated  base  as  pre- 
existing in  the  original  material  only  when  it  produces 
symptoms  identical  with  those  caused  by  the  substance  from 
which  it  is  extracted.  There  can  be  no  doubt  that  by  this 
method  many  ptoma'iues  would  be  decomposed.  With  it 
Ehrenberg  obtained  from  poisonous  sausage  only  inert 
bases,  and  tyrotoxicon,  the  ptomaine  of  poisonous  cheese, 
is  decomposed  both  by  heat  and  the  hydrogen  sulphide 
employed.  The  origin  of  the  ptomaines  possessing  a  mus- 
carine-like  action  discovered  by  Brieger  has  been  ques- 
tioned by  Gram,  who  states  that  when  the  lactate  of  choline, 
an  inert  substance  which  is  widely  distributed  both  in  plants 
and  animals,  is  heated,  it  is  converted  into  a  poison  with 
such  an  action. 


CHAPTER  IX. 

METHODS    OF    ISOLATING   THE    BACTERIAL    PROTEIDS. 

Hankin  employed  the  following  process  in  preparing 
his  anthrax  proteid.  : 

"  The  cultures  are  made  in  0.1  per  cent.  Liebig's  extract 
of  meat  solution,  to  which  some  fibrin  is  added.  The  Lie- 
big's extract  is  very  difficult  to  sterilize,  and  must  be  heated 
for  two  or  three  hours  in  the  steam  sterilizer  on  two  or 
three  successive  days.  The  fibrin  must  be  added  only  after 
this  has  been  done,  and  then  the  flask  is  re-sterilized  by 
repeated  heating  to  boiling-point,  for  a  short  time  only  on 
each  occasion.  If  the  fibrin  were  added  at  first  it  would 
be  decomposed  by  the  prolonged  boiling.  By  the  above 
method  this  only  occurs  to  a  slight  degree,  a  mere  trace  of 
peptone  being  present  in  the  sterilized  culture-fluid.  After 
sterilizing,  this  is  inoculated  with  the  blood  of  an  animal 
dead  of  anthrax,  and  kept  at  the  ordinary  temperature. 
The  anthrax  forms  a  typical  growth  on  the  masses  of  fibrin, 
and  samples  of  the  liquid  removed  on  successive  days  show 
a  gradual  increase  in  the  strength  of  their  biuret  reaction. 
After  about  a  week  the  liquid  is  filtered  and  the  albumose 
extracted.  The  reason  for  not  keeping  the  flask  at  a  tem- 
perature of  37°  is  that  the  albumose  is  gradually  decom- 
posed into  peptone  by  the  anthrax  ferment  present,  and 
this  change  takes  place  more  rapidly  at  the  higher  tempera- 
ture. For  instance,  I  have  found  scarcely  a  trace  of  albu- 
mose in  a  culture  which  had  been  kept  at  37°  for  a  week, 
and  which  gave  a  strong  biuret  reaction.  The  albumose 
is  separated  from  the  culture-liquid  thus  prepared  by  satu- 
ration with  ammonium  sulphate.  It  is  better  to  acidulate 
it  slightly  by  adding  a  little  acetic  acid.  The  bulky  pre- 
cipitate of  albumose  which  then  appears  is  filtered  off,  and 
the  salt  separated  from  it  by  dialysis.    An  excess  of  thymol 


172  BACTERIAL    POISON'S. 

must  be  added  at  this  stage  to  prevent  putrefaction,  or  the 
dialysis  cau  be  carried  ou  in  a  current  of  water  which  is 
warmed  to  from  45°  to  50°  C,  at  which  temperature  the 
growth  of  microorganisms  is  inhibited.  After  dialyziug 
for  twenty-four  hours  or  more  the  greater  part  of  the  salt 
will  have  vanished,  and  the  albumose  will  be  found  in 
solution  in  a  considerable  quantity  of  water  which  will  not 
have  passed  through  the  parchment.  It  is  now  necessary 
merely  to  concentrate  the  solution  and  precipitate  the 
albumose  by  the  addition  of  alcohol.  In  my  earlier  ex- 
periments this  was  accomplished  by  evaporating  in  vacuo 
at  a  temperature  of  45°  to  48°.  When  at  length  the  liquid 
has  been  reduced  to  a  few  cubic  centimetres  it  is  poured 
into  alcohol,  and  the  precipitated  albumose  is  filtered  off, 
washed  with  the  same  reagent  (alcohol),  and  dried. 

"  Evaporating  in  vacuo  is  a  long  and  tedious  process, 
and  it  requires  a  somewhat  complicated  apparatus.  When 
it  is  used  for  pathogenic  albumoses  there  is  always  a  risk 
of  the  temperature  employed  destroying  or  diminishing 
their  physiological  properties.  Further,  if  the  albumose 
is  allowed  to  evaporate  to  dryness,  it  may  be  difficult  to 
make  it  pass  into  solution  again.  To  avoid  these  difficul- 
ties I  have  designed  a  method  of  concentrating  such  solu- 
tions which  is  less  objectionable.  It  depends  on  the  prin- 
ciple that,  if  alcohol  and  water  are  placed  on  opposite. sides 
of  a  membrane,  the  water  rapidly  dialyzes  through  to  mix 
with  the  alcohol,  while  only  traces  of  alcohol  pass  through 
to  mix  with  the  water.  Consequently,  if  a  watery  solu- 
tion of  albumoses  is  dialyzed  against  alcohol,  the  solution 
diminishes  in  bulk  and  is  rapidly  concentrated,  owing  to 
the  passage  of  the  water  through  the  membrane. 

"  My  modus  operandi  is  to  place  the  dilute  albumose 
solution  in  a  parchment  sausage  skin  which  is  immersed  in 
a  foot  glass  full  of  methylated  spirit.  The  spirit  can  be 
changed  after  some  hours  if  it  is  desired  to  prolong  the 
process ;  but  this  is  not  usually  necessary.  In  this  way  I 
have  been  able  to  bring  400  c.c.  of  albumose  solution  down 
to  100  c.c.  in  the  course  of  a  single  night,  at  the  ordinary 
temperature,  without  risk  to  the  albumose  or  trouble  to 


ISOLATING    THE    BACTERIAL    PROTEIDS.      173 

myself!  The  concentrated  solution  is  then  poured  into 
absolute  alcohol,  which  precipitates  the  albumose  and  re- 
moves any  impurities  that  might  be  derived  from  the 
methylated  spirit.  This  prolonged  treatment  with  alcohol 
will  tend  to  remove  any  free  ptomaines  or  other  substances 
soluble  in  alcohol.  Peptones  and  salts  present  in  the  cul- 
ture liquid  remained  for  the  most  part  in  solution  when 
the  albumose  was  precipitated  with  (NH4)2S04.  No  soluble 
proteids  (except  traces  of  peptone)  were  present  in  the  cul- 
ture medium." 

Ordinarily  the  bacterial  proteids  are  isolated  by  preci- 
pitation with  absolute  alcohol,  re-solution  in  water  and  re- 
precipitation  with  alcohol.  However,  as  has  been  stated, 
Tizzoni  and  Cattani  find  that  strong  alcohol  destroys  the 
activity  of  the  poison  of  their  tetanus  germ.  The  method 
employed  in  obtaining  the  bacterial  cellular  proteids  has 
already  been  given  (see  page  130). 


CHAPTER    X. 

THE   IMPORTANCE    OF  PTOMAINES  TO  THE   TOXICOLOGIST. 

The  presence  in  the  cadaver  of  substances  which  give 
not  only  the  general  alkaloidal  reactions  but  respond  to 
some  of  the  tests  which  have  hitherto  been  considered 
characteristic  of  individual  vegetable  alkaloids,  must  be 
of  the  greatest  importance  to  toxicologists.  The  possi- 
bility of  mistaking  putrefactive  for  vegetable  alkaloids 
should  always  be  borne  in  mind  by  the  chemist  in  making 
his  medico-legal  investigations.  On  the  other  hand,  as  we 
have  seen  in  preceding  chapters,  cases  of  poisoning  by 
ptomaines  sometimes  terminate  fatally,  and  in  such  in- 
stances the  chemist  should  not  be  satisfied  with  determin- 
ing the  absence  of  mineral  and  vegetable  poisons,  but 
should  strive  to  detect  in  the  food  or  in  the  dead  body 
positive  evidence  of  the  presence  of  the  putrefactive 
alkaloid. 

We  will  give  a  brief  account  of  those  cases  in  which 
putrefactive  substances  have  been  found  to  resemble  in 
their  reactions  the  vegetable  alkaloids. 

Conhne-like  Substances. — The  most  celebrated  case 
in  which  a  substance  giving  reactions  similar  to  those  of 
coniine  has  been  found,  was  the  Brandes-Krebs  trial, 
which  took  place  in  Braunschweig  in  1874.  From  the 
uudecomposed  parts  of  the  body  two  chemists  obtained, 
in  addition  to  arsenic,  au  alkaloid  which  they  pronounced 
coniine.  This  substance  was  referred  to  Otto  for  further 
examination.  Otto  reported  that  the  substance  was 
neither  coniine  nor  nicotine,  nor  any  vegetable  alkaloid 
with  which  he  was  acquainted.  Otto  converted  the  sub- 
stance into  an  oxalate,  dissolved  it  in  alcohol,  evaporated 
the  alcohol,  dissolved  the  residue  in  water,  rendered  this 


CONIINE-LIKE    SUBSTANCES.  175 

solution  alkaline  with  potash,  then  extracted  the  base  with 
petroleum  ether.  On  evaporation  of  the  petroleum  ether 
the  alkaloid  appeared  as  a  bright  yellow  oil,  which  had  a 
strong,  unpleasant  odor,  quite  different,  however,  from 
that  of  coniine.  It  was  strongly  alkaline  and  had  an 
intensely  bitter  taste.  At  ordinary  temperature  it  was 
volatile.  From  its  aqueous  solution  it  was  precipitated 
by  the  chlorides  of  gold,  platinum,  and  mercury.  In 
these  reactions  it  resembled  nicotine,  from  which  it  differed 
in  the  double  refractive  and  crystalline  character  of  its 
hydrochloride.  With  an  ethereal  solution  of  iodine  this 
substance  did  not  give  the  Roussin  test  for  nicotine,  but. 
instead  of  the  long  ruby-red  crystals  there  appeared  small, 
dark-green,  needle-shaped  crystals. 

This  substance  was  found  to  be  highly  poisonous.  Seven 
centigrammes  injected  subcutaueously  into  a  large  frog  pro- 
duced instantaneous  death,  and  forty-four  milligrammes 
given  to  a  pigeon  caused  a  similar  result.  On  account  of  its 
poisonous  properties  the  jury  of  medical  experts  decided 
that  the  substance  was  a  vegetable  alkaloid.  Otto  says 
that  this  decision  astounded  the  chemists. 

Brouardel  and  Bodtmy  found  in  the  body  of  a  woman, 
who  had  died,  after  suffering,  with  ten  other  persons,  from 
choleraic  symptoms  from  eating  of  a  stuffed  goose,  a  base 
which  gave  the  odor  of  coniine  and  the  same  reactions  with 
gold  chloride  and  iodine  in  potassium  iodide,  etc.,  as 
coniine.  The  same  base  was  found  in  the  remainder  of 
the  goose.  But  it  did  not  give  a  red  coloration  with  the 
vapor  of  hydrochloric  acid,  and  it  did  not  form  butyric 
acid  on  oxidation,  and  although  it  was  poisonous,  it  did 
not  produce  in  frogs  the  symptoms  of  coniine  poisoning. 

Selmi  repeatedly  found  coniine-like  substances  in  de- 
composing animal  tissue.  By  distilling  an  alcoholic  extract 
from  a  cadaver,  acidifying  the  distillate  with  hydrochloric 
acid,  evaporating,  treating  the  residue  with  barium  hydrate 
and  ether,  aud  allowing  the  ether  to  evaporate  spontane- 
ously, he  obtained  a  residue  of  volatile  bases,  the  greater 
part  of  which  consisted  of  trimethylamine.  After  remov- 
ing the  trimethylamine,  the  residue  had  the  odor  of  the 


176  BACTERIAL    POISONS. 

urine  of  the  mouse.  Later,  Selmi  obtained  an  unmistak- 
able coniine  odor  from  a  chloroform  extract  of  the  viscera 
of  a  person  who  had  been  buried  six  months,  and  in  an- 
other case  ten  months  after  burial.  Two  or  three  drops  of 
an  aqueous  solution  of  the  alkaline  residue  of  the  chloro- 
form extract  allowed  to  evaporate  on  a  glass  plate  gave  on0 
such  a  penetrating  odor  that  Selmi  was  compelled  to  with- 
draw from  close  proximity  to  the  substance.  The  odor 
imparted  to  his  hands  in  testing  the  substance  with  the 
general  alkaloidal  reagents  remained  for  half  an  hour. 
This  volatile  base  seemed  to  be  formed  by  the  spontane- 
ous decomposition  of  other  ptomaines. 

An  aqueous  solution  of  a  ptomaine  obtained  by  Selmi 
by  extraction  with  ether  according  to  the  Stas-Otto 
method  from  the  undecomposed  parts  of  a  cadaver  had 
no  marked  odor,  but  after  having  been  kept  for  a  long  time 
in  a  sealed  tube  it  not  only  gave  off  a  marked  coniine 
odor,  but  the  vapor  turned  red  litmus-paper  blue.  Again, 
the  sulphate  of  a  ptomaine  obtained  from  putrid  egg- 
albumin,  on  standing  formed  in  two  layers,  one  of  which 
was  a  golden-yellow  liquid,  which  on  being  treated  with 
barium  hydrate  gave  off  ammonia,  and  later,  the  odor  of 
coniine.  Since  butyric  and  acetic  acids  were  formed  by 
the  oxidation  of  this  base,  Selmi  concluded  that  he  had 
real  coniine  or  methylconiiue,  and  that  it  was  formed  by 
the  oxidation  of  certain  fixed  ptomaines,  or  by  the  action 
of  different  amido  bases  on  volatile  fatty  acids.  There- 
fore Selmi  believed  in  the  spontaneous  origin  of  coniine 
or  closely  allied  bases  in  putrid  matter,  also  in  the  exist- 
ence of  a  "  cadaveric  coniine." 

The  substauce  which  was  found  by  Sonnenschein  in  a 
criminal  trial  in  East  Prussia,  and  which  was  believed  by 
that  chemist  to  be  the  alkaloid  of  the  water  hemlock  (cicuta 
virosa),  is  thought  by  Otto,  Husemanjst,  and  others,  to  be 
a  cadaveric  coniine.  Otto  says  that  the  symptoms  re- 
ported in  the  case  were  not  those  of  either  coniine  or 
cicuta.  Sonnenschein  obtained  the  base  six  weeks  after 
the  exhuming  of  the  body,  which  had  been  buried  three 
months.     The  base  had  the  odor  of  coniine,  the  taste  of 


A    NICOTINE-LIKE    SUBSTANCE.  177 

tobacco,  gave  with  potassium  bichromate  and  sulphuric 
acid  the  odor  of  butyric  acid,  and  behaved  with  reagents 
like  couiine. 

Husemann  states  that  at  present  it  is  very  difficult,  if 
not  impossible,  for  the  chemist  to  state  with  certainty  that 
he  has  detected  true  coniine  iu  the  dead  body.  The  symp- 
toms and  the  post-mortem  appearances  must  conform  with 
those  induced  by  the  vegetable  alkaloid.  The  analysis 
must  be  made  before  decomposition  sets  in,  and  the  amount 
of  the  base  found  must  be  sufficient  for  physiological  ex- 
periments to  be  made  with  it. 

A  Nicotine-like  Substance. — "Wolckenhaar  ob- 
tained from  the  decomposed  intestines  of  a  womau,  who 
had  been  dead  six  weeks,  by  extraction  with  ether  from  an 
alkaline  solution,  a  base  which  bore  a  close  resemblance  to 
nicotine.  The  base  was  fluid,  at  first  yellow,  but  on  being 
exposed  to  the  air,  brownish-yellow.  It  was  strongly  alka- 
line in  reaction  and  gave  off  an  odor  resembling  nicotine, 
but  stronger,  not  ethereal,  but  benumbing  and  similar  to 
that  of  fresh  poppy-heads.  It  was  soluble  in  all  propor- 
tions iu  water,  and  the  solutions,  which  did  not  become 
cloudy  on  the  application  of  heat,  did  not  taste  bitter,  but 
were  slightly  pungent.  The  peculiar  odor  did  not  disap- 
pear on  saturating  the  base  with  oxalic  acid.  The  hydro- 
chloride was  yellow,  like  varnish,  had  a  strong  odor,  and 
became  moist  on  exposure  to  the  air.  Under  the  micro- 
scope it  showed  no  crystals,  differing  in  this  respect  from 
nicotine  hydrochloride.  It  differed  from  nicotine  also  in 
its  reactions  with  potassio-bismuthic  iodide,  gold  chloride, 
iodine  solution,  mercuric  chloride,  and  platinum  chloride. 
It  also  failed  to  give  the  Roussin  test  for  nicotine.  More- 
over, it  could  not  be  identified  with  trimethylamine,  spar- 
teine, mercurialine,  lobeline,  or  other  fluid  and  volatile 
bases. 

The  studies  of  Rorsch  and  Fassbender  (page  28),  of 
Schwanert  (page  28),  of  Liebermann  (page  30),  and 
of  Selmi  (page  81),  have  already  been  referred  to  in  a 
preceding  chapter. 


178  BACTERIAL    POISONS. 

Strychnine-like  Substances. — In  a  criminal  prose- 
cution at  Veroua,  Ciotta  obtained  from  the  exhumed,  but 
only  slightly  decomposed  body,  an  alkaloid  which  gave  a 
crystalline  precipitate  with  iodine  in  hydriodic  acid,  a  red 
coloration  with  hydriodic  acid,  and  a  color  test  similar  to 
that  of  strychnine  with  sulphuric  acid  and  potassium 
bichromate,  and  with  other  oxidizing  agents.  This  sub- 
stance was  strongly  poisonous,  but  did  not  produce  the 
tetanic  convulsions  which  are  characteristic  of  strychnine. 
Ciotta  pronounced  this  substance  as  probably  identical 
with  strychnine.  Portions  of  the  body  were  subsequently 
submitted  to  Selmi  for  his  opiniou.  Selmi  found  that 
the  substance  which  gave  the  color-reaction  was  not  crys- 
talline, and  that  there  was  only  "  the  presumption  of  a 
bitter  taste  to  it,"  while  one  part  of  strychnine  in  40,000 
parts  of  water  is  intensely  bitter.  Selmi  also  held  that 
many  ptomaines  give  reactions  similar  to  strychnine  with 
iodine  in  hydriodic  acid  and  with  hydriodic  acid.  He 
also  held  that  its  physiological  properties  were  such  that  it 
could  not  be  strychnine.  This  substance  could  hardly 
have  been  aspidospermine,  which  reacts  with  sulphuric 
acid  and  potassium  bichromate  similarly  to  strychnine,  be- 
cause quebracho  bark,  in  which  this  alkaloid  is  found,  was 
not  at  that  time  used  as  a  medicine  or  known  in  Italy. 

Ptomaines  giving  reactions  similar  to  those  of  strych- 
nine, and  also  causing  tetanic  spasms,  have  been  found  in 
Italy  in  decomposed  corn-meal.  Selmi  obtained  one  of 
these  substances,  but  found  that  it  differed  from  strychnine 
inasmuch  as  it  could  not  be  extracted  with  ether. 

Lombroso  has  named  the  poisonous  substance  found  in 
decomposed  corn-meal  pellagroceine,  but  this  is  really  a 
mixture  of  ptomaines,  some  of  which  produce  narcosis  and 
paralysis,  and  others  produce  the  symptoms  of  nicotine 
poisoning  instead  of  the  spasms  caused  by  strychnine. 

A  Morphine-like  Substance. — In  the  Souzogna 
trial,  at  Cremona,  Italy,  the  experts  seem  to  have  con- 
founded a  ptomaine  with  morphine.  This  substance  was 
not  removed  from  either  alkaline  or  acid  solutions  with 


DIGITALINE-LIRE    SUBSTANCES.  179 

ether,  but  could  be  extracted  with  amylic  alcohol.  It 
reduces  iodic  acid,  but  iu  its  other  reactions,  as  well  as  in 
its  physiological  properties,  it  bore  no  resemblance  to 
morphine.  In  frogs  it  arrested  the  heart  in  systole,  which 
is  said  never  to  happen  in  poisoning  with  morphine.  It 
failed  to  give  both  the  ferric  chloride  and  the  Pellagri 
tests  for  morphine. 

In  the  same  body  there  was  found  a  substance  which 
was  extracted  from  alkaline  solutions  with  ether,  and  .which 
gave,  with  hydrochloric  acid  and  a  few  drops  of  sulphuric 
acid,  on  the  application  of  heat,  a  reddish  residue  similar 
to  that  obtained  by  the  same  reagents  with  codeine,  but  iu 
its  other  reactions  it  did  not  resemble  this  alkaloid. 

Ateopine-like  Substances. — Many  investigators  have 
found  products  of  putrefaction  which  in  their  mydriatic 
properties  resemble  atropine  and  hyoscyamine.  To  this 
class  belongs  the  substance  observed  by  Zuelzer  and  Son- 
nenschein.  It  was  removed  from  alkaline  solutions  by 
ether,  and  formed  microscopic  crystals,  an  aqueous  solution 
of  which,  when  applied  to  the  conjunctiva,  produced  a 
mydriatic  effect,  and,  when  administered  internally,  in- 
creased the  action  of  the  heart  and  arrested  the  movements 
of  the  intestines.  Moreover,  with  certain  alkaloidal  re- 
agents, such  as  platinum  chloride,  it  resembled  atropine. 
But  when  heated  with  sulphuric  acid  and  oxidizing  agents 
it  did  not  give  the  odor  of  blossoms  (Reuss's  test).  How- 
ever, Selmi  found  ptomatropines  which  with  sulphuric 
acid  and  oxidizing  agents  did  give  the  blossom  odor  as  dis- 
tinctly as  the  vegetable  atropine.  These  putrefactive  bases 
also  developed  this  odor  spontaneously  after  standing  for 
two  or  three  days,  and  this  does  not  happen  with  atropine. 
The  odor  was  produced  with  the  ptomatropines  by  nitric 
and  sulphuric  acids,  both  in  the  cold  and  on  the  applica- 
tion of  heat,  while  these  acids  in  the  cold  do  not  produce 
the  odor  with  atropine. 

Digitaline-like  Substances. — Elsewhere  we  have 
referred  to  the  discovery  of  a  ptomaine  belonging  to  this 


180  BACTERIAL    POISONS. 

class  by  Rorsch  and  Fassbender  (see  page  28).  Trot- 
tarelli  obtained  a  similar  substance  from  the  brain  of  a 
man  in  whose  abdominal  viscera  he  could  find  no  poison. 
The  sulphate  of  this  base  gave  on  evaporation  an  aromatic- 
smelling  and  astringent-ta-ting  residue.  It  became  purple 
with  sulphuric  acid  alone,  and  dark  red  with  hydrochloric 
and  sulphuric  acids.  On  frogs  this  ptomaine  showed  no 
toxic  effect. 

A  Veratrlne-like  Substance. — Brouardel  and 
Boutmy  obtained  from  a  corpse  which  had  lain  in  water 
for  eighteen  months,  and  a  large  portion  of  which  had 
changed  into  adipocere,  a  ptomaine  resembling  veratrine. 
It  was  removed  from  alkaline  solutions  by  ether.  On 
being  heated  with  sulphuric  acid  it  became  violet.  With 
a  mixture  of  sulphuric  acid  and  barium  peroxide  it  be- 
came, in  the  cold,  brick-red ;  and,  on  being  heated,  violet. 
With  boiling  hydrochloric  acid  it  took  on  a  cherry-red 
coloration.  However,  it  differed  from  veratrine,  inasmuch 
as  it  reduced  ferric  salts  instantly,  and  when  injected  into 
frogs  subcutaneously  it  did  not  induce  in  them  the  spas- 
modic muscular  contractions  characteristic  of  veratrine. 

Bechamp  obtained  by  the  Stas-Otto  method  from  the 
products  of  the  pancreatic  digestion  of  fibrin  an  alkaloid 
body  which  gave  with  sulphuric  acid  a  beautiful  carmine- 
red,  similar  to  that  given  with  veratrine.  By  digesting  this 
substauce  with  gastric  juice,  and  again  extracting,  he 
obtained  a  body  which  behaved  with  sulphuric  acid  similar 
to  curarine. 

A  Delphinine-like  Substance. — In  1870,  General 
Gibbone,  an  Italian  of  prominence,  died  suddenly.  His 
servant  was  accused  of  having  poisoned  him.  Two  chem- 
ists of  some  reputation  reported  the  presence  of  delphinine 
in  the  viscera.  It  seemed  somewhat  improbable  that  the 
servant  should  know  anything  of  so  rare  a  substance,  or 
that  he  should  have  been  able  to  obtain  it.  However,  two 
or  more  varieties  of  staphisagria  grow  in  Southern  Italy, 
and  it  was  possible  that  the  servant  had  used  some  prepara- 


A    COLCHICINE-LIKE    SUBSTANCE.  181 

tion  made  by  himself  from  the  plant.  The  supposed  alka- 
loid was  given  to  Selmi,  of  Bologna,  for  further  study.  It 
was  removed  from  alkaline  solutions  by  ether.  When 
heated  with  phosphoric  acid  it  became  red,  and  when 
brought  in  contact  with  concentrated  sulphuric  acid,  red- 
dish-brown. In  these  tests  the  substance  resembled  delph- 
inine,  but  with  sulphuric  acid  and  bromine  water,  also 
with  Frohde's  reagent,  the  colorations  characteristic  of 
the  vegetable  alkaloid  failed  to  appear.  Moreover,  Selmi 
showed  that  delphinine  gave  the  following  reactions,  to 
which  the  suspected  substance  did  not  respond  :  (1)  Delph- 
inine dissolved  in  ether,  and  treated  with  a  freshly  prepared 
ethereal  solution  of  platinic  chloride,  gives  a  white,  floccu- 
lent  precipitate  which  is  insoluble  in  an  equal  volume  of 
absolute  alcohol.  (2)  Delphinine  gives  precipitates  with 
auro-sodium  hyposulphite,  and  with  a  sulphuric  acid  solu- 
tion of  cupro-sodium  hyposulphite,  the  latter  precipitate 
being  soluble  in  an  excess  of  the  reagent. 

Finally,  Ciaccia  and  Vella  showed  that  while  delph- 
inine arrests  the  heart  of  the  frog  in  diastole,  the  suspected 
substance  arrests  it  in  systole. 

A  Colchicine-like  Substance. — Baumert  found  in 
a  suspected  case  of  poisoning,  twenty-two  months  after 
death,  a  substance  which  gave  many  of  the  reactions  for 
colchicine.  It  was  extracted  from  acid  solutions  with  ether, 
to  which  it  imparted  a  yellow  color.  On  evaporation  of 
the  ether  a  yellow,  amorphous  substance  remained,  and 
this  dissolved  in  warm  water  with  yellow  coloration.  It 
could  be  extracted  from  acid  solutions  also  by  chloroform, 
benzol,  and  amylic  alcohol,  but  not  by  petroleum  ether.  It 
was  removed  with  much  more  difficulty  from  alkaline 
solutions. 

All  the  extracts  were  yellow,  and  left  on  evaporation  a 
feebly  alkaline,  markedly  bitter,  sharp-tasting,  amorphous, 
yellow  residue,  which  dissolved  in  water  and  dilute  acids 
incompletely,  forming  a  resin.  When  this  resin  was  dis- 
solved in  dilute  sodium  hydrate,  and  the  solution  rendered 

9 


182  BACTERIAL    POISONS. 

acid  by  sulphuric  acid,  the  same  reactions  were  obtained  as 
with  the  original  extract. 

With  phosphomolybdic  acid,  phosphotungstic  acid,  potas- 
sio-bismuthic  iodide,  potassio-rnercuric  iodide,  iodine  in 
potassium  iodide,  tannic  acid  and  gold  chloride,  this  sub- 
stance gave  the  same  reactions  which  were  obtained  by 
parallel  experiments  with  genuine  colchicine ;  thus,  the 
tannic  acid  precipitates  were  both  soluble  in  alcohol,  and 
the  precipitates  with  phosphomolybdic  acid  in  both  cases 
became  blue  on  the  addition  of  ammonium  hydrate. 

Concentrated  sulphuric  and  dilute  nitric  and  hydrochloric 
acids  dissolved  the  supposed  colchicine  with  yellow  colora- 
tion. Strong  nitric  acid  (1.4  sp.  gr.)  colored  the  substance 
dirty  red,  scarcely  to  be  called  a  violet.  When  the  sub- 
stance was  purified  as  much  as  possible,  this  color  became 
a  beautiful  carmine-red.  The  addition  of  water  changed 
the  red  into  yellow,  and  caustic  soda  produced  a  dark,  dirty 
orange. 

In  general,  in  the  above-mentioned  reactions,  the  putre- 
factive product  agreed  with  the  real  colchicine,  but  the 
former  gave  precipitates  with  picric  acid  and  platinum 
chloride,  while  the  latter  gives  no  precipitates  with  these 
reagents. 

In  1886,  Zeisel  proposed  the  following  test  for  colchi- 
cine :  When  a  hydrochloric  acid  solution  of  the  alkaloid  is 
boiled  with  ferric  chloride,  it  becomes  green,  sometimes 
dark-green  and  cloudy.  Now,  if  the  fluid  be  agitated  with 
chloroform,  the  chloroform  will  sink,  taking  up  the  coloring 
matter,  and  appearing  brownish,  granite-red  or  dark,  and 
the  supernatant  fluid  clears  up  without  becoming  wholly 
colorless. 

Baumert  applied  this  test  to  both  colchicine  and  the 
putrefactive  product.  To  from  two  to  five  cubic  centi- 
metres of  the  suspected  solution  in  a  test-tube,  he  added 
from  five  to  ten  drops  of  strong  hydrochloric  acid  and 
from  four  to  six  drops  of  a  ten  per  cent,  solution  of  ferric 
chloride,  then  heated  the  mixture  directly  over  a  small 
flame  until  it  was  evaporated  to  half  its  volume  or  less.  In 
the  presence  of  one  milligramme  of  colchicine  the  originally 


MORPHINE.  183 

bright-yellow  solution  became  gradually  olive-green,  and, 
on  further  concentration,  dark-green  and  cloudy.  Then, 
on  shaking  the  fluid  with  chloroform,  admitting  as  much 
air  as  possible,  the  chloroform  subsided,  having  a  ruby-red 
color  if  as  much  as  two  milligrammes  of  colchicine  were 
present,  and  a  bright  yellow  if  only  one  milligramme,  and  the 
supernatant  fluid  became  of  a  beautiful  olive-green.  When 
ether,  petroleum  ether,  benzol,  carbon  disulphide,  or  amylic 
alcohol  was  substituted  for  the  chloroform,  the  coloration 
did  not  appear.  From  this  Baumert  infers  that  the  red 
coloring  matter  is  either  only  soluble  in  chloroform,  or  that 
it  is  not  formed  until  the  chloroform  is  added. 

Baumert  found  this  test  of  great  value  in  deciding 
whether  or  not  the  substance  which  he  found  was  colchi- 
cine. The  putrefactive  product  did  not  respond  to  the 
test. 

Some  of  this  substance  was  sent  to  Brieger,  who  de- 
cided that  it  was  not  a  base,  but  a  peptone-like  substance. 
It  was  also  found  to  be  inert  physiologically. 

Before  these  investigations  were  made  by  Baumert, 
Liebermann  had  found  the  same  or  a  similar  colchicine- 
like  substance  in  the  cadaver.  His  description  differed 
from  that  of  Baumert  only  in  regard  to  the  taste  of  the 
substance,  Liebermann  having  failed  to  observe  any 
marked  taste  in  the  substance  which  he  found,  while,  as  has 
been  stated,  Baumert  reported  a  distinctly  bitter  taste. 

A  colchicine-like  substance  has  been  found  in  beer,  and 
it  has  been  suggested  that  it  was  this  which  the  above- 
mentioned  toxicologists  found  in  the  bodies  which  they 
examined,  but  Liebermann  states  that  the  man  whose 
body  he  examined  had  been  a  total  abstainer  from  beer. 

Tamba  compared  the  reactions  of  ptomaines  obtained 
from  putrid  sausage  with  similar  reactions  of  various  alka- 
loids, and  then  ascertained  the  effect  upon  the  alkaloidal 
reactions  by  mixing  the  alkaloids  with  the  ptomaines.  His 
results  are  as  follows  : 

Morphine. — Ptomaines  are  colored  yellow  with  nitric 
acid ;    reddish-yellow    with   concentrated    sulphuric   acid ; 


184  BACTERIAL    POISONS. 

blue,  violet,  then  green  with  Frohde's  reagent ;  yellow 
when  evaporated  with  concentrated  sulphuric  acid,  then 
treated  with  hydrochloric  acid  aud  decomposed  with  sodium 
bicarbonate.  The  ptomaines  reduce  ferric  chloride,  but  not 
iodic  acid.  With  sugar  and  concentrated  sulphuric  acid, 
they  give  a  yellow  coloration. 

Mixtures  of  the  ptomaines  and  morphine  give  absolutely 
characteristic  reactions  for  morphine  with  sugar  and  sul- 
phuric acid,  the  violet  coloration  appearing  distinctly ;  and 
by  evaporation  on  the  water-bath  with  sulphuric  acid,  addi- 
tion of  hydrochloric  acid  and  decomposition  with  sodium 
bicarbonate,  the  violet  color  appearing.  Iodic  acid  is  re- 
duced by  morphine  in  the  presence  of  ptomaines,  only 
when  the  ptomaines  are  present  in  minute  quantity. 

The  other  reactions  for  morphine  are  not  applicable  in 
the  presence  of  ptomaines. 

Strychnine. — The  characteristic  color  reaction  for  this 
alkaloid,  with  potassium  bichromate  and  sulphuric  acid,  is 
not  affected  by  the  presence  of  ptomaines.1 

Brucine. — The  nitric  acid  reaction  for  brucine  is  not 
affected  by  ptomaines.  On  the  other  hand,  the  reaction 
with  sulphuric  and  nitric  acids,  in  which  a  red  coloration 
is  obtained,  is  scarcely  visible  in  the  presence  of  ptomaines. 
The  action  of  mercuric  nitrate  and  heat  on  brucine,  by  which 
a  violet  coloration  is  produced,  is  not  destroyed  by  the 
presence  of  ptomaines. 

Veratrine. — The  characteristic  coloration  of  veratrine 
by  concentrated  sulphuric  acid  is  not  influenced  by  pto- 
maines. The  same  is  true  of  the  cherry-red  coloration  with 
concentrated  hydrochloric  acid.  On  the  contrary,  the  action 
of  sugar  and  sulphuric  acid  on  veratrine  is  without  result 
in  the  presence  of  ptomaines. 

Atropine. — The  deep  violet  coloration  produced  by 
fuming  nitric  acid,  subsequent  concentration,  and  the  addi- 

1  In  contradiction  to  this,  see  page  178. 


DELPHININE.  185 

tion  of  alcoholic  potassium  hydrate,  is  not  affected  by  the 
presence  of  ptomaines.  On  the  other  hand,  the  character- 
istic odor  produced  by  the  action  of  sulphuric  acid  and 
heat  on  atropine  is  scarcely  recognizable  when  ptomaines 
are  present. 

Earceine. — The  blood-red  color  produced  by  concen- 
trated sulphuric  acid  fails  in  the  presence  of  ptomaines. 

Colchicine. — Fuming  nitric  acid  colors  the  ptomaines 
reddish-yellow,  but  the  violet  coloration  of  colchicine  with 
nitric  acid  appears  in  well-defined  form,  even  in  the  pres- 
ence of  ptomaines.  The  other  reactions  for  colchicine  are 
valueless  when  ptomaines  are  present. 

Codeine. — The  blue  coloration  of  codeine  with  concen- 
trated sulphuric  acid  holds  good  when  ptomaines  are  present. 
The  same  is  true  of  the  reaction  with  sulphuric  acid,  heat, 
and  the  subsequent  addition  of  nitric  acid.  Frohde's  re- 
agent fails  with  codeine  when  mixed  with  ptomaines,  inas- 
much as  the  bluish  coloration  rapidly  passes  into  a  brown. 

Aconitine. — Phosphoric  acid  and  concentrated  sulphuric 
acid  are  without  reaction  on  the  alkaloid  when  mixed  with 
ptomaines. 

Picrotoxine. — The  reducing  action  of  picrotoxine  on 
alkaline  copper  sulphate  solution  is  seriously  affected  by  the 
presence  of  ptomaines.  The  same  is  true  of  other  tests  for 
this  poison. 

Delphinine. — The  reaction  of  delphinine  with  sulphuric 
acid  and  bromine  water,  as  well  as  the  one  with  Frohde's 
reagent,  is  so  much  influenced  by  the  presence  of  ptomaines 
that  the  alkaloid  cannot  be  recognized. 

These  results  are  to  be  accepted  with  caution,  as  it  is  not 
reasonable  to  suppose  that  all  ptomaines  will  affect  the  test 
for  the  vegetable  alkaloids  in  the  same  manner  or  to  the 


186  BACTEEIAL    POISONS. 

same  degree.  Moreover,  there  is  no  proof  that  Tamba 
worked  with  pure  ptomaines. 

Tamba  has  also  proposed  to  separate  vegetable  from 
putrefactive  alkaloids  by  adding  to  ethereal  solutions  of 
mixtures  an  equal  volume  of  a  saturated  ethereal  solution 
of  oxalic  acid,  and  allowing  to  stand,  when  the  oxalates  of 
the  vegetable  alkaloids  will  separate  in  crystalline  form, 
and  the  oxalates  of  the  ptomaines  will  remain  in  solution. 
In  other  words,  the  oxalates  of  the  vegetable  alkaloids  are 
insoluble  in  ether,  while  the  oxalates  of  the  putrefactive 
alkaloids  are  soluble  in  ether.  But,  in  contradiction  to  this, 
Bocklisch  states  that  the  oxalate  of  cadaverine  is  insoluble 
in  ether. 

The  most  important  work  which  the  toxicologist  is  called 
upon  to  do  at  present  is  to  isolate  and  identify  beyond  all 
question  the  bacterial  poisons.  This  work  has  become  im- 
portant on  account  of  the  frequent  occurrence  of  poisoning 
from  articles  of  infected  food. 


CHAPTER  XI. 

CHEMISTEY   OF   THE   PTOMAINES. 

The  basic  substances  described  in  the  following  pages 
are  arranged,  so  far  as  possible,  in  the  regular  natural  order. 
An  inspection  of  the  list  of  these  bases  will  show  the  remark- 
able fact  of  the  predominancy  of  the  amine  type.  Almost 
two-thirds  of  the  known  ptomaines  contain  only  C,  H,  and 
N,  and  represent  simple  ammonia  substitution  compounds. 
Of  the  oxygenated  bases,  all  of  those  whose  constitution  is 
known  possess  the  trimethylamine  molecule  as  their  basic 
constituent,  and  it  is  quite  probable  that  most,  if  not  all,  of 
the  remaining  ptomaines  will  be  found  to  possess  the  same 
or  a  similar  basic  nucleus. 

It  will  be  seen,  furthermore,  that  a  very  large  number  of 
the  ptomaines  described  possess  little  or  no  toxic  action, 
and  are,  therefore,  physiologically  inert.  It  would  seem, 
as  Brieger  has  already  pointed  out,  that  a  certain  quantity 
of  oxygen  is  necessary  to  the  formation  of  poisonous  bases. 
A  free  supply  of  oxygen,  on  the  other  hand,  invariably 
yields  non-toxic  ptomaines.  The  poisonous  bases  begin  to 
appear  on  about  the  seventh  day  of  putrefaction,  and  in 
turn  disappear  if  this  is  allowed  to  go  on  for  a  considerable 
period  of  time. 

Methyl  amine,  CH3.NH2. — This  is  the  simplest  organic 
base  that  is  formed  in  the  process  of  putrefaction.  It  is 
ammonia  in  which  one  atom  of  hydrogen  has  been  replaced 
by  the  methyl  radical.  It  occurs  in  herring-brine  (Tol- 
lens,  1866  ;  Bocklisch,  1885)  ;  in  decomposing  herring, 
twelve  days  in  spring  (Bocklisch)  ;  in  pike,  six  days  in 
summer  (Bocklisch)  ;  in  haddock,  two  months  at  a  low 
temperature  (Bocklisch)  ;  in  the  fermentation  of  choline 
chloride  (Hasebroek).     Brieger   has   shown  it  to  be 


188  BACTERIAL    POISONS. 

present  in  cultures  of  comma  bacillus  on  beef-broth  which 
were  kept  for  six  weeks  at  37°-38°.  Ehrenberg  re- 
ported its  possible  presence  in  poisonous  sausage,  and  ob- 
tained it  by  growing  a  bacillus  from  this  source  on  intes- 
tines (1887).  In  Brieger's  method,  methylamine  is  found 
both  in  the  mercuric  chloride  precipitate  and  filtrate.  The 
mercury  double  salt  is  readily  soluble  in  water,  and  can 
thus  be  separated  from  any  accompanying  cadaverine  or 
putrescine.  Methylamine  is  an  inflammable  gas  of  strong 
ammoniacal  odor,  and  burning  with  a  yellow  flame.  It  is 
readily  soluble  in  water,  and  its  solutions  give  reactions 
similar  to  those  of  ammonia.  Its  salts  are,  as  a  rule,  also 
soluble  in  both  water  and  alcohol. 

The  Hydrochloride,  CH3NH2.HC1,  crystallizes  in 
large  deliquescent  plates.  On  being  heated  with  alkali,  it 
gives  off  the  odor  of  methylamine. 

~  The  Platinochloride,  (CH3.NH2.HCl)2PtCl4  (Pt  = 
41.31  per  cent.),1  yields  hexagonal  plates  which  usually 
occur  heaped  up  in  several  layers.  It  is  soluble  in  about 
fifty  parts  of  water  at  ordinary  temperature,  and  can  be 
readily  recrystallized  from  hot  water.  It  is  insoluble  in 
absolute  alcohol  and  in  ether. 

The  Aurochloride,  CH3.NHa.HCl.AuCl3  +  H20, 
forms  prisms,  which  are  readily  soluble  in  water.  There 
is  also  a  readily  soluble  picrate. 

Methylamine  does  not  possess  any  toxic  action,  even  when 
given  in  fairly  large  doses.  This  physiological  indifference 
is  shared  by  nearly  all  the  monamines  and  diamines  that 
have  been  obtained  among  the  products  of  putrefaction. 

Dimethylamine,  (CH3)2.NH,  has  been  found  in  putre- 
fying gelatin,  ten  days  at  35°  (Brieger,  1885);  in  yeast 
decomposing  in  covered  vessels  for  four  weeks  during  sum- 
mer (Brieger)  ;  in  decomposing  perch,  six  days  in  summer 
(Bocklisch)  ;  and  in  herring-brine  (Bocklisch:,  1886).  It 
has  been  found  in  poisonous  sausage,  and  in  cultures  of  a 

1  The  percentages  given  in  the  following  pages  are  calculated  from  Au= 
196.64  (Kriiss),  Pt  =  194.46  (Seubert),  CI  =  35.37,  0  =15.96, 


CHEMISTEY    OF    THE    PTOMAINES.  189 

bacillus  obtained  from  this  source,  on  liver  and  intestines 
(Ehrenberg,  1887).  It  is  also  formed,  together  with 
trimethylamine,  when  neuridine  hydrochloride  is  distilled 
with  sodium  hydrate  (Brieger,  I.,  23).  It  occurs  in  the 
mercuric  chloride  precipitate  as  well  as  nitrate.  From 
cadaverine  it  can  be  separated  by  platinum  chloride,  since 
cadaverine  platinoehloride  is  difficultly  soluble  in  cold  water, 
and  recrystallizes  from  hot  water,  whereas  the  dimethyl- 
amine  double  salt  remains  in  the  mother-liquor.  In  like 
manner  it  can  be  separated  from  neuridine.  From  choline 
it  can  be  isolated  by  recrystallizing  the  mercuric  chloride 
precipitate  from  hot  water. 

The  free  base  is  a  gas  at  ordinary  temperature,  but  can 
be  condensed  to  a  liquid  which  boils  at  8°  -9°.  The 
hydrochloride,  (CH3)2.NH.HC1,  crystallizes  in  needles, 
which  deliquesce  on  exposure  to  air  and  are  soluble  in  abso- 
lute alcohol  (Brieger,  I  ,  56).  It  is  insoluble  in  absolute 
alcohol  (Bocklisch)  but  soluble  in  chloroform  (Behrend), 
and  can  then  be  separated  from  methylamine  hydrochloride, 
which  is  insoluble  in  chloroform. 

The  Platinochloride,  [(CH3)2.NH.HCl]2PtCl4,  (Pt 
=»  39.00  per  cent.),  crystallizes  in  long  needles,  which  are 
easily  soluble  in  hot  water,  less  soluble  in  cold  water.  Some- 
times it  forms  orange-yellow  plates  or  prisms,  or  else  small 
needles. 

The  Aurochloride,  (CH3)2.NH.HCl.AuCl3,  forms 
needles  (Bocklisch),  or  large  yellow  monoclinic  plates 
(Hjortdahl),  which  are  insoluble  in  absolute  alcohol. 

Trimethylamine,  C3H9N"  =  (CH3)3N,  has  been  known 
for  a  long  time  to  occur  in  animal  and  vegetable  tissues. 
Dessaignes  showed  its  presence  in  leaves  of  Chenopodium 
(1851),  in  the  blood  of  calves  (1857),  and  later  in  human 
urine.  It  has  been  obtained  from  ergot  (Secale  cornutum) 
by  Walz  (1852)  and  Brieger  (1886) ;  from  herring-brine 
by  Wertheim,  Winkles,  Tollens,  and  Bocklisch. 
In  these  substances,  with  the  exception  of  herring-brine,  it 
probably  does  not  exist  pre-formed,  but  is  rather  a  product 
of  the  method  employed  for  its  isolation.     In  fact,  Brieger 


190  BACTERIAL    POISONS. 

has  shown  that  it  does  not  exist  in  ergot,  but  is  formed  at 
the  expense  of  the  choline  present,  which,  on  distillation 
with  potash,  decomposes  and  yields  trimethylamine  and 
glycol.     Thus : 

C2H4OH.N(CH3)3.OH  =  N(CH3)3  +  C2H4(OH)2. 

It  is  also  formed  when  betaine  and  neuridine  are  distilled 
with  potash.  It  may  have  a  similar  origin  in  most  of  the 
other  cases,  since  choline  is  now  known  to  be  widely  dis- 
seminated in  plants  and  animals,  either  as  such  or  as  a 
constituent  of  the  more  complex  lecithin.  Trimethylamine 
has  been  found  in  the  putrefaction  of  yeast  (Hesse,  1857  ; 
Muller,  1858) ;  in  cheese  after  six  weeks  in  midsummer 
(Brieger)  ;  in  human  liver  and  spleen  after  from  two  to 
seven  days  (Brieger)  ;  in  perch  after  six  days  in  mid- 
summer (Bockllsch)  ;  in  mussel  (Mytilus  edulis)  after  six- 
teen days  (Brieger)  ;  in  putrefying  braius  after  from  one 
to  two  months,  and  in  fresh  brains  (Guareschi  and 
Mosso) ;  in  cultures  of  the  Streptococcus  pyogenes  on  beef- 
broth,  bouillon,  meat  extract,  and  blood-serum,  and  from 
cultures  of  the  comma  bacillus  (Brieger).  It  has  also 
been  found  in  cod-liver  oil.  Ehrenberg  (1887)  reports 
its  presence  in  considerable  quantity  in  poisonous  sausage, 
and  in  cultures  of  a  bacillus,  isolated  from  this,  grown  on 
liver,  intestines,  and  meat  bouillon. 

Trimethylamine  is  found  both  in  the  mercuric  chloride 
precipitate  and  nitrate.  It  remains  in  the  mother-liquor 
from  which  cadaverine,  neuridine,  and  dimethylamine  pla- 
tinochlorides  have  crystallized.  If  an  aqueous  solution  of 
mercuric  chloride  is  used  as  the  precipitant,  the  trimethyl- 
amine will  be  found  almost  entirely  in  the  filtrate,  from 
which  it  can  be  obtained  after  removal  of  the  mercury  by 
evaporating  the  nitrate  to  dryness,  extracting  with  alcohol, 
and  treating  the  solution  thus  obtained  with  alcoholic  pla- 
tinum chloride. 

The  free  base  is  a  liquid  possessing  a  strong,  fish-like 
odor.  Its  boiling-point  is  9.3°.  It  is  strongly  alkaline  in 
reaction  and  freely  soluble  in  water. 

The  Hydrochloride,  (CH3)3N.HC1,  is  deliquescent  and 


CHEMISTRY    OF    THE    PTOMAINES.  191 

freely  soluble  in  water  and  alcohol.  Heated  to  285°  it 
decomposes.  With  alkalies  it  gives  off  the  odor  of  the 
free  base. 

The  Platinochloride,  [(CH3)3N.HCl]2PtCl4  (Pt  = 
36.92  per  cent.),  is  soluble  in  hot  water,  from  which,  on 
cooling,  it  reciy stalli zes  in  orange-red  octahedra  or  needles, 
which  do  not  lose  water  when  heated  at  100°— 110°  (Bock- 
lisch). 

The  Aurochloride,  (CH3)3N.HCl.AuCl3  (An  =  49.39 
per  cent.),  is  easily  soluble,  and  hence  can  be  separated  from 
choline  aurochloride,  which  is  difficultly  soluble.  Similarly, 
this  base  can  be  separated  from  ammonia  by  the  use  of  gold 
chloride. 

Trimethylamine  is  not  a  strong  poison,  since  very  large 
doses  of  it  must  be  given  in  order  to  bring  out  any  physio- 
logical disturbances. 

Ethylamine,  C2H5.NH2,  is  formed  in  putrefying  yeast 
(Hesse,  1857) ;  in  wheat  flour  (Sullivan,  1858) ;  and  also 
in  the  distillation  of  beet-sugar  residues. 

It  is  a  strongly  ammoniacal  liquid  boiling  at  18.7°,  and 
is  miscible  with  water  in  every  proportion.  Like  the  other 
amines,  it  is  combustible.  It  possesses  strong  basic  prop- 
erties, and  is  capable  of  expelling  ammonia  from  its  salts  in 
a  manner  analogous  to  the  action  of  the  fixed  alkalies. 

The  Hydrochloride,  C2H5.lSrH2.HCl,  forms  deliques- 
cent plates,  which  melt  at  76°-80°.  It  is  readily  soluble 
in  water  and  alcohol. 

The  Platinochloride,  (C2H5.NH2.HCl)2PtCl4,  forms 
orange-yellow  rhombohedra  (Weltzien),  or  hexagonal- 
rhombohedral  crystals  (Topsoe). 

The  Aurochloride,  C2H5.NH2.HC1.  AuC13,  forms  gold- 
yellow  monoclinic  prisms,  readily  soluble  in  water. 

With  picric  acid  it  forms .  short  brown  prisms,  not  very 
soluble  in  water. 

Diethylamine,  C4HnN  =  (C2H5)2NH,  has  been  ob- 
tained by  Bocklisch  from  pike  which  were  allowed  to 
putrefy  for  six  days  in  summer  ;  and  by  growing  a  bacillus 


192  BACTERIAL    POISONS. 

obtained  from  poisonous  sausage  on  intestines  and  on  meat 
bouillon  (Ehrenberg,  1887). 

It  is  an  inflammable  liquid  which  boils  at  57.5°,  possesses 
strong  basic  properties,  and  is  soluble  in  water. 

The  Hydrochloride,  (C2H5)2NH.HC1,  crystallizes  in 
needles  (Bocklisch)  ;  in  long  needles  and  prisms  from 
absolute  alcohol ;  in  plates  from  ether-alcohol.  These  are 
not  deliquescent  and  are  easily  soluble  in  Avater  and  in 
chloroform  ;  rather  difficultly  in  absolute  alcohol.  Heated 
with  sodium  hydrate  it  gives  off  alkaline  vapors.  From  an 
alcoholic  solution  it  is  precipitated  by  addition  of  alcoholic 
mercuric  chloride.  The  mercury  double  salt  is  difficultly 
soluble  in  hot  water,  from  which  it  recrystallizes  on  cooling. 

The  Platinochloride,  [(C2H5)2.NH.HCl]2PtCl4,  crys- 
tallizes in  orange-yellow  monoclinic  crystals,  which  are 
easily  soluble  in  water. 

The  Aurochloride,   (C2HS)2.NH.HC1. AuCl3   (Au  = 

47.71  per  cent.),  forms  trimetric  crystals  (Topsoe),  which 

are  difficultly  soluble  (Bocklisch).    It  melts  at  about  165°. 

With  picric  acid  it  forms  an  easily  soluble  picrate  (Lea). 

Triethylamine,  C6H15N  =  (C2H5)3N,  was  obtained 
by  Brieger  (1885)  from  haddock  which  were  exposed  for 
five  days  in  an  open  vessel  during  summer.  He  obtained 
it  by  distilling  with  potash,  after  removal  of  platinum  by 
hydrogen  sulphide,  the  mother  liquor  from  which  neuridine, 
the  base  C2H8N2,  muscarine,  and  gadinine  had  successively 
crystallized  (see  Gadinine).  It  has  also  been  found  by 
Bocklisch  (1886)  in  putrid  pike,  and  by  Ehrenberg 
(1887).  The  latter  obtained  it  from  cultures  of  a  bacillus, 
found  in  poisonous  sausage,  and  grown  on  meat  bouillon. 

The  free  base  is  oily  in  character  and  possesses  an  am- 
moniacal  odor.  It  is  but  slightly  soluble  in  water,  and 
boils  at  89°-89.5°. 

The  Platinochloride,  [(C2H5)3KHCl]2PtCl4  (Pt  = 
31.84  per  cent.),  crystallizes  in  needles  which  are  readily 
soluble  in  water. 

With  mercuric  chloride  the  aqueous  solution  gives  no 
precipitate. 


CHEMISTRY    OF    THE    PTOMAINES.  193 

With  picric  acid  it  yields  yellow  needles  which  are  but 
slightly  soluble  in  cold  water. 

Propylamine,  C3H7.]SrH2?  is  isomeric  with  trimethyl- 
amine,  and  can  therefore  be  easily  confounded  with  that 
base.  There  are  two  propylamines  possible  represented 
by  the  formulae  CH3.CH2.CH2.NH2  and  (CH3)2.CH.NH2. 
The  former,  or  the  normal  compound,  boils  at  47°-48°, 
whilst  the  latter,  or  iso-propylamine,  boils  at  31.5°.  Both 
are  liquids  possessing  an  ammoniacal,  fish-like  odor.  They 
form  crystalline  salts  ;  the  hydrochlorides  melt  respectively 
at  155°-158°,  and  at  139.5°. 

Iso-propylamine  (?)  has  been  found  among  the  distilla- 
tion products  of  the  vinasse  of  beet-root  molasses.  Propyl- 
amine has  been  obtained  by  Brieger  (1887)  from  cultures 
of  the  bacteria  of  human  feces  on  gelatin.  Schwanert 
has  isolated  from  the  organs  of  a  cadaver  a  basic  substance 
which  was  said  to  possess  an  odor  similar  to  propylamine. 

Butylamine,  C4HnN,  was  obtained  by  Gautier  and 
Mourgues  (1888)  in  cod-liver  oil.  It  forms  a  colorless, 
mobile,  alkaline  liquid,  the  boiling-point  of  which  they 
found  to  be  86°  at  760  mm.  It  absorbs  carbonic  acid  from 
the  air  and  readily  forms  salts.  The  platinochloride  forms 
golden-yellow  plates  which  are  quite  soluble. 

In  animals  it  produces  an  increase  in  the  function  of  the 
skin  and  kidneys,  and  in  large  doses  fatigue,  stupor,  and 
vomiting. 

Iso-amylamine,  C5H13N  =  (CH3)2.CH.CH2.CH2.NH2, 
has  been  obtained  by  Limpricht  in  the  distillation  of  horn 
with  potash  ;  it  also  occurs  in  the  putrefaction  of  yeast 
(Muller,  Hesse,  1857) ;  and  in  cod-liver  oil  (Gautier 
and  Mourgues,  1888),  where  it  constitutes  nearly  one- 
third  of  the  bases  present. 

It  is  a  colorless,  strongly  alkaline  liquid,  possessing  an 
odor  which  is  not  disagreeable.  At  the  ordinary  pressure 
it  boils  at  97°-98°. 

The   hydrochloride   forms  deliquescent  crystals,  which 


194  BACTEKIAL    POISONS. 

have  a  bitter,  disagreeable  taste.  The  platinoehloride  crys- 
.tallizes  in  golden-yellow  slender  plates,  which  are  very 
soluble  in  boiling  water.  The  base  is,  according  to  Gau- 
tier  and  Mourgues,  identical  with  that  obtained  by  treat- 
ing iso-amylcarbimide  with  potash. 

It  is  a  very  active  poison,  producing  rigor,  convulsions, 
and  death.  Four  milligrammes  produces  death  in  a  green- 
finch in  three  minutes. 

Caproylamine  (Hexylamine),  C6H15N,  has  been 
found  to  occur  by  Hesse  (1857)  in  the  putrefaction  of 
yeast.  Hager  isolated  from  some  putrid  material  what  he 
thought  to  be  a  mixture  of  amylamine  and  caproylamine, 
and  named  it  septicine. 

Hexylamine  was  found,  in  small  quantity,  in  cod-liver 
oil  by  Gautier  and  Mourgues,  and  according  to  these 
authors  it  resembles  amylamine  in  its  action,  but  is  less 
toxic. 

Tetanotoxine,  C5HnN",  (?)  was  obtained  by  Brieger 
(1886)  as  one  of  the  products  of  the  growth  of  the  tetanus 
microbe  on  beef-broth  or  on  brain-broth.  It  has  also  been 
obtained  by  Kitasato  and  Weyl  (1890)  from  pure  cultures 
of  the  tetanus  bacillus,  kept  eight  days  at  36°.  For  its 
isolation  see  Tetanine,  and  Ber.  19,  3120.  It  is  tetanizing 
in  its  action,  produces  first  tremor,  then  paralysis  and  vio- 
lent convulsions.  It  forms  an  easily  soluble  gold  double 
salt  which  melts  at  130°.  The  platinoehloride  is  difficultly 
soluble,  and  decomposes  at  240°.  The  hydrochloride  is 
crystalline,  and  is  readily  soluble  in  alcohol  and  in  water. 
It  melts  at  about  205°.  From  warm  alcohol  it  crystallizes 
in  flat,  pointed  plates. 

Spasmotoxine,  a  base  of  as  yet  unknown  composition, 
produces  in  animals  violent  clonic  and  tonic  convulsions. 
It  was  obtained  by  Brieger  (1887)  from  cultures  of  the 
tetanus  germ  on  beef-broth. 


CHEMISTEY    OF    THE    PTOMAINES.  195 

Another  toxine  was  obtained  by  Brieger  (1887)  in  cult- 
ures of  the  tetanus  microbe  which  produced  complete  tetanus, 
salivation,  and  tear-secretion.  In  its  composition  it  is  prob- 
ably a  diamine.  The  platinochloride  forms  plates  which 
begin  to  decompose  at  240°.  The  hydrochloride  is  very 
deliquescent.  Gold  chloride  and  picric  acid  form  very 
soluble  compounds.  Besides  these  three  bases  he  isolated 
another  toxic  substance,  tetanine,  and  a  base  (see  under 
Tetaniue). 

Dihydrolutidine,  C7HnN,  was  found  in  cod-liver  oil 
by  Gautier  and  Mourgues  (1888).  It  is  the  first  known 
hydrolutidine.  It  is  a  colorless,  somewhat  oily,  very  alka- 
line and  caustic  liquid,  the  odor  of  which  is  sharp,  but 
somewhat  agreeable  when  dilute.  It  absorbs  carbonic  acid 
from  the  air,  darkens  and  thickens  ;  is  feebly  soluble  in 
water,  and  boils  at  199°  at  760  mm.  pressure.  The  salts 
are  bitter  to  the  taste. 

The  hydrochloride  crystallizes  in  a  confused  mass  of 
needles  or  in  plates.  The  nitrate  reduces  silver  nitrate — a 
property  of  all  hydropyridine  bases  (Hofmann).  The 
sulphate  forms  fine  stellate  deliquescent  needles. 

The  platinochloride  is  readily  precipitated  from  concen- 
trated solutions  as  a  canary-yellow  precipitate.  From 
warm  solutions  it  crystallizes  in  lozenge-shaped  plates  which 
are  often  imbricated.  On  boiling  with  water  it  loses  hydro- 
chloric acid  and  forms  (C7HuNCl)2PtCl2,  which  possesses  a 
lighter  color,  is  more  soluble  than  the  normal  salt,  and  crys- 
tallizes confusedly. 

The  aurochloride  crystallizes  in  needles  which  form  fan 
or  lozenge-shaped  masses.  It  is  scarcely  altered  even  in  hot 
water. 

The  Iodomethylate,  C7HuN.CH3I,  is  obtained  by  mix- 
ing, in  the  cold,  the  base  and  methyl  iodide.  The  colorless 
compound  thus  obtained  is  soluble  in  water  and  in  alcohol, 
and  possesses  a  disagreeable,  somewhat  nauseating  odor. 
Treated  with  potash  it  yields  a  colorless,  aromatic,  very 
alkaline  oil. 

The  base  on  oxidation  with  boiling  potassium  perman- 


196  BACTERIAL    POISONS. 

ganate  yields  an  acid,  C7H7N02,  and  from  this  fact  the 
discoverers  conclude  that  the  base  is  a  dihydro-dimethyl- 
pyridine,  C5H4(CH3)2NH. 

Physiological  Action. — It  is  moderately  poisonous.  In 
small  doses  it  diminishes  the  general  sensibility ;  in  larger 
doses  it  produces  trembling,  especially  of  the  head ;  pro- 
found depression  alternating  with  periods  of  extreme  ex- 
citement ;  paralysis  of  the  posterior  limbs,  and  death. 

A  Base,  C8H1XN,  isomeric,  but  not  identical,  with  alde- 
hyde-collidine,  was  obtained  by  Nencki  as  early  as  1876, 
by  allowing  a  mixture  of  200  grammes  of  pancreas  and  600 
grammes  of  gelatin  in  ten  litres  of  water  to  putrefy  for  five 
days  at  40°.  The  method  used  by  Nencki  for  its  isola- 
tion is  as  follows  :  The  fluid  mass  was  distilled  with  sul- 
phuric acid,  to  drive  off  the  volatile  acids,  then  rendered 
alkaline  with  barium  hydrate,  and  again  distilled.  The 
distillate  was  received  in  dilute  hydrochloric  acid,  and  on 
evaporation  gave  a  crystalline  residue  of  ammonium  chlo- 
ride, and  of  a  salt  which  formed  in  long  rhombic  plates. 
The  latter  were  separated  from  the  ammonium  salt  by  abso- 
lute alcohol.  The  free  base  was  obtained  from  the  salt  by 
treating  it  with  sodium  hydrate,  and  extracting  the  solution 
with  ether. 

This  compound,  as  already  stated,  is  isomeric  with  colli- 
dine,  and  also  with  O.  de  Coninck's  base,  with  which  it  is 
possibly  identical.  The  latter,  however,  will  be  described 
separately. 

The  free  base  is  oily  in  character,  and  possesses  a  peculiar, 
not  unpleasant  odor.  It  readily  absorbs  carbonic  acid  gas 
from  the  air,  forming  after  a  time  a  lamellar,  crystalline 
mass  of  the  carbonate.  The  salt  of  this  base  on  heating 
gives  off  an  oil  which  burns  with  a  smoky  flame,  and  pos- 
sesses an  odor  similar  to  that  of  xylol  or  cumol.  Nencki 
was  therefore  at  first  of  the  opinion  that  the  ptomaine  was 

an  aromatic  base,  probably  an  isophenyl-ethylamine  of  the 

sr\  it 

following  composition:    C6H5  —  CH\^yj3.    He  supposed 


CHEMISTRY    OF    THE    PTOMAINES.  197 

that  it  was  formed  from  the  putrefaction  of  tyrosin,  accord- 
ing to  the  following  equation  : 

C,HuN08  =  C8HUK  +  C02  +  0. 

We  know  that  tyrosin  does  split  up,  on  being  heated  to 
270°,  into  carbonic  acid  and  oxyphenyl-ethylamine,  thus  : 

°eH<cI2.OH.N1rrCOOH  =  C6H/g|2CH2NH2  +  c02 

In  1883  Erlenmeyer  and  Lipp  observed  that  phenyl- 
a-amido-propionic  acid  (phenyl-alanine),  on  dry  distillation, 
decomposed  with  the  formation,  among  other  products,  of  a 
base  having  the  composition  C8Hu]Sr.  This  base  was  found 
to  be  identical  with  phenyl-ethylamine,  C6H5.CH2.OH2.NH2, 
and  in  its  properties  and  composition  it  resembles  Nencki's 
base.  Recently  (1889),  Nencki  has  taken  up  a  similar 
view  in  regard  to  the  nature  of  this  base,  and  now  regards 
it  as  possessing  the  formula  just  given — that  it  is  phenyl- 
ethylamine.  He  regards  phenyl-amido-propionic  acid — one 
of  the  three  aromatic  nuclei  contained  in  the  albumin  mole- 
cule— as  the  source  of  this  base.  From  the  fact  that  phenyl- 
a-amido-propionic  acid  is  a  well-known  putrefactive  product, 
it  would  seem  that  Nencki's  base  may  arise  either  from  the 
putrefactive  decomposition  of  that  acid,  or  from  the  splitting 
up  of  the  acid  as  a  consequence  of  the  method  employed  in 
isolating  the  base.  The  latter  would  seem  to  be  the  most 
probable  explanation  of  the  genesis  of  this  base,  inasmuch 
as  Brieger,  by  using  his  method  for  the  isolation  of  pto- 
maines, has  not  been  able  to  obtain  it  from  putrid  gelatin. 

The  Platinochloride,  (C8HnN.HCl)2PtCl4  (Pt  = 
29.89  per  cent.),  is  readily  soluble  in  hot,  and  but  slightly 
soluble  in  cold  water,  and  can  be,  therefore,  recrystallized 
from  water.  It  forms  beautiful  flat  needles.  On  dry 
heating  it  gives  off  an  oil  which  possesses  an  odor 
resembling  very  much  that  of  xylol  or  cumol,  and  burns 
with  a  smoky  flame.  This  distinguishes  Nencki's  base 
from  collidine,  since  the  platinochloride  of  the  latter  does 
not  show  this  behavior. 

Nencki  also  obtained  from  putrid  gelatin,  under  certain 


198  BACTERIAL    POISONS. 

ill-defined  conditions,  especially  when  no  glycocoll  was 
present,  a  basic  product  which  gave,  with  sulphuric  acid, 
large  lamellar  crystals.  The  free  base  forms  a  thick  color- 
less syrup,  possessing  a  nauseous,  bitter  taste.  It  did  not 
become  crystalline  even  after  standing  some  time.  Unlike 
the  base  C8H1VN",  it  is  not  volatile,  and  is,  therefore,  obtained 
on  evaporation  of  the  acidulated  solution  after  previous 
removal  of  the  volatile  bases  by  distillation  with  baryta. 

A  Base,  C8HuN,  isomer  of  collidine  and  of  the  preceding 
base,  with  which  it  is  possibly  identical,  was  obtained  by  0. 
de  Conlnck  (1888)  in  the  later  stages  of  putrefaction  of 
sea-polyps  (poulpes  marins).  It  forms  a  yellowish,  rather 
mobile  liquid,  possessing  a  strong  benumbing  (vireuse)  odor, 
and  is  but  slightly  soluble  in  water.  It  is  soluble  in  methyl 
and  ethyl  alcohol,  ether  and  acetone.  Its  density  is  0.9865. 
When  dried  over  potash  it  boils  at  202°  without  undergoing 
decomposition.  On  exposure  to  the  air  it  becomes  brown, 
hydrates  rapidly,  and  the  boiling-point  is  then  lowered.  It 
has  not  been  noticed  to  absorb  carbonic  acid  from  the  air. 
It  resembles  some  of  the  bases  obtained  from  Dippel's  oil. 
The  salts  are  in  general  less  stable  than  those  of  the  pyri- 
dine bases,  and  in  this  respect  it  approaches  the  dihydro- 
pyridine  bases. 

The  Hydrochloride,  C8IInN.HCl,  forms  white  or 
slightly  yellowish  radiate  masses  which  are  deliquescent 
and  very  soluble  in  water.  The  hydrobromide,  C8HnN. 
HBr,  resembles  it,  but  is  less  deliquescent  and  a  trifle  less 
soluble  in  cold  water. 

The  Platinochloride,  (G\HuKHCl)2PtCl4,  is  a  dark 
orange-colored  powder,  which  is  insoluble,  or  almost  so,  in 
cold  water,  and  is  a  rather  stable  compound.  Boiling 
water  and  water  at  80°  decompose  it  into  hydrochloric 
acid  and  (C8HnNCl)2PtCl2,  which  is  a  light-brown  powder, 
insoluble  in  cold,  scarcely  so  in  hot  water. 

The  Aurochloride,  C8HuN.HC1.AuC13,  forms  a  light 
yellow  precipitate.  It  is  quite  stable  in  cold,  but  very  un- 
stable in  hot  or  even  warm  water.  It  cannot  be  modified 
by  withdrawal  of  hydrochloric  acid. 


CHEMISTRY    OF    THE    PTOMAINES.  199 

It  forms  two  compounds  with  mercuric  chloride.  (C8HU 
N.HCl)2HgCl2  crystallizes  in  small  white  needles,  which 
are  slightly  soluble  in  water  and  in  dilute  alcohol,  insoluble 
in  absolute  alcohol,  and  on  exposure  to  moist  air  undergo 
change.  The  second  compound,  2(C8HnN.HCl).3HgCl2,  is 
obtained  by  adding  an  excess  of  concentrated  mercuric 
chloride  to  a  concentrated  solution  of  the  hydrochloride. 
It  forms  slightly  yellow,  somewhat  longer  needles  which 
are  insoluble  in  the  principal  solvents,  and  are  likewise 
changed  by  atmospheric  humidity. 

The  Iodomethylate,  CgHnlSr.CH^I,  is  formed  by 
mixing  solutions  of  the  base  and  methyl  iodide  in  absolute 
ether.  It  is  deposited  as  a  network  of  fine  white  needles, 
which  are  but  slowly  altered  in  the  air,  and  are  soluble  in 
absolute  alcohol.  This  solution  on  the  addition  of  a  little 
potash  assumes  a  dark-red  color,  which  is  heightened  by 
the  addition  of  a  little  hydrochloric  or  acetic  acid,  and 
destroyed  by  ammonia  without  any  resultant  fluorescence. 
Warmed  with  excess  of  moist  solid  potash  it  becomes 
garnet-red  in  color  and  gives  off  an  odor  resembling  that 
of  the  dihydropyridines.  It  thus  behaves  the  same  as  the 
pyridine  iodomethylates. 

On  oxidation  with  potassium  permanganate  it  yields  an 
acid  which  melts  at  229°-230°,  and  begins  to  sublime  at 
150°.  It  presents  all  the  characteristics  of  nicotinic  acid, 
C6H5N02,  which  is  formed  as  the  result  of  oxidation  of 
nicotine.  With  hydrochloric  acid  it  forms  the  compound, 
C6HVN"02.HC1.  With  copper  acetate  it  forms  a  salt ;  this, 
distilled  with  lime,  yields  a  substance  which  on  boiling 
with  platinum  chloride  and  water  forms  the  compound 
(C5H5NCl)2.PtCl2.  This  same  substance  forms  an  iodo- 
methylate, which  in  alcoholic  solution  gives,  on  addition  of 
potash,  the  characteristic  reaction  of  pyridine  bases. 

The  base,  C8Hnl^,  therefore  yields  pyridine  and  nico- 
tinic acid. 

A  Base,  C8H13N,  was  obtained  by  Gautier  and  Etard 
(1881)  from  the  chloroformic  extracts  (see  method,  page  164) 
from  putrefying  mackerel,  as  well  as  from  the  decomposing 


200  BACTERIAL    POISONS. 

flesh  of  the  horse  and  ox.  It  is  regarded  by  these  authors 
as  a  constant  and  definite  product  of  the  bacterial  fermenta- 
tion of  albuminoid  substances,  but  this  view  is  hardly  justi- 
fiable, inasmuch  as  the  base  has  not  been  found  by  other 
investigators.  It  is  accompanied  by  the  base  C17H38N4 
(page  228).  Nencki  (1882)  asserted  the  identity  of  this 
base  with  the  one  which  he  had  isolated  in  1876,  and  to 
which  he  had  ascribed  the  formula  C8HUN.  On  the  other 
hand  Gautier  and  Etard  consider  their  base  to  be  iden- 
tical with  the  hydrocollidine  obtained  by  Cahours  and 
Etard  by  the  action  of  selenium  on  nicotine. 

The  free  base  is  an  alkaline,  almost  colorless,  oily  liquid, 
possessing  a  penetrating  odor  resembling  that  of  syringa. 
It  is  volatile  without  decomposition,  and  boils  at  about  205°, 
while  hydrocollidine  boils  at  210°.  Its  density  at  zero  is 
1.0296.  When  exposed  to  the  air  it  oxidizes  slowly,  be- 
comes brown  and  viscous,  and  at  the  same  time  absorbs 
carbonic  acid.  It  differs  from  a  collidine  in  possessing  a 
strong  reducing  action,  since  both  the  gold  and  platinum 
double  salts  become  reduced  on  heating,  and  even  in  the 
cold. 

The  Hydrochloride,  C8H13lSr.IICl,  is  very  soluble  in 
water  and  in  alcohol,  and  usually  forms  fine  needles  re- 
sembling snow  crystals.  It  is  neutral  in  reaction  and  pos- 
sesses a  bitter  taste.  In  the  presence  of  an  excess  of  acid 
it  reddens  and  resinifies. 

The  Platinochloride,  (C8H13N.HCl)2PtCl4  (Pt  = 
29.7  per  cent.),  is  of  a  light  yellow,  flesh-color,  crystalline, 
and  but  slightly  soluble.  It  dissolves  on  warming,  and  re- 
crystallizes  in  bent  needles. 

The  Aurochloride  is  rather  soluble,  and  becomes 
slowly  reduced  in  the  cold  ;  rapidly  on  warming. 

Physiological  Action. — This  isomer  of  hydrocollidine 
is  strongly  poisonous.  Even  so  small  a  dose  as  0.0017 
gramme  of  the  hydrochloride  produced,  when  injected  under 
the  skin  of  a  bird,  marked  unsteadiness  of  gait,  followed  by 
paralysis  of  the  extremities,  and  finally  death.  The  pupils 
are  normal  and  the  heart  stops  in  diastole.  Larger  doses 
(0.007  gramme)  cause  at  first  vomiting  and  staggering, 


CHEMISTRY    OF    THE    PTOMAINES.  201 

which  soon  give  way  to  a  condition  of  exaltation.  Toward 
the  end  tetanic  convulsions  set  in,  followed  by  almost  com- 
plete paralysis. 

A  Base,  C9H13N,  isomeric  with  parvoline,  has  been  ex- 
tracted by  Gautier  and  Etard  (1881)  from  decomposing 
mackerel  and  horseflesh.  The  method  employed  by  these 
chemists  for  its  isolation  is  given  on  page  164.  The  iden- 
tity of  this  base  with  the  synthetic  parvoline,  obtained  by 
Waage  by  heating  ammonia  with  propionic  aldehyde  in  a 
sealed  tube  at  200°,  cannot  be  considered  to  be  definitely 
settled,  although  an  apparent  identity  exists  in  regard  to 
their  boiling-points.  Thus,  the  synthetic  parvoline  boils  at 
193°-196°,  while  Gautier  and  Etard  assign  to  their 
base  a  boiling-point  a  little  below  200°.  Further  investi- 
gation is  necessary  to  decide  upon  the  question  of  the  iden- 
tity of  this  base  with  parvoline,  or  of  the  ptomaine  C8H13N 
with  hydrocollidine. 

The  free  base  is  an  oily,  amber-colored  liquid,  possessing 
the  odor  of  hawthorn  blossoms.  It  is  slightly  soluble  in 
water  ;  very  soluble  in  alcohol,  in  ether,  and  in  chloroform. 
Its  boiling-point,  as  stated  above,  is  a  trifle  below  200°. 
Like  the  bases  C8H13N  and  C10H15N  it  becomes  brown  and 
soon  resinifies  on  exposure  to  air. 

The  Platinochloride,  (C9H13N.HCl)2PtCl4  (Pt  = 
28.65  per  cent.),  is  slightly  soluble,  crystalline,  and  flesh 
colored ;  exposed  to  the  air  it  soon  becomes  pink. 

The  Aurochloride  is  quite  soluble. 

A  Base,  C10H15N,  was  isolated  by  Guareschi  and 
Mosso  (1883)  from  ox-blood  fibrin  which  had  been  allowed 
to  putrefy  for  five  months.  In  1887  it  was  re-obtained  from 
putrid  fibrin  by  Guareschi,  who  this  time  ascribed  to  it  the 
formula  C10H13N.  In  1886  Oechsner  de  Coninck  found 
it  among  the  basic  products  formed  in  the  putrefaction  of 
the  jelly-fish  (poulpes  marins,  Hugounenq,  page  21).  The 
method  used  for  its  extraction  was  that  of  Gautier  and 
Etard  (see  page  164).  It  forms  a  brownish  oil  of  strong 
alkaline  reaction,  which  soon  resinifies.     It  possesses  an 


202  BACTERIAL    POISOKS. 

unpleasant,  weak  pyridine  or  coniine  odor,  and  is  but 
slightly  soluble  in  water;  soluble  in  ether  and  in  chlo- 
roform. 

In  regard  to  the  constitution  of  this  ptomaine  we  know 
nothing,  but  from  its  physical  characters  it  would  seem  to 
possess  a  pyridine  nucleus.  It  is  isomeric  with  corindine, 
a  homologue  of  parvoline  and  collidine,  which  has  been 
obtained  from  coal-tar. 

For  the  behavior  of  the  hydrochloride  to  alkaloidal  re- 
agents, see  Table  I. 

The  Hydrochloride,  C10H15N.HC1,  crystallizes  in 
colorless  cholesterine-like  plates  which  are  somewhat  deli- 
quescent. 

The  Platinochloride,  (C10H15N.HCl)2PtCl4  (Pt  = 
27.52  per  cent.),  forms  a  light  flesh-colored,  crystalline  pre- 
cipitate, and  is  insoluble  in  water,  alcohol,  and  ether.  It 
does  not  resinify,  and  is  stable  at  100°. 

Physiological  Action. — This  ptomaine  resembles  curara, 
although  it  is  by  no  means  as  strong.  0.012  gramme  of 
the  free  base  produced  in  a  frog  dilatation  of  the  pupil  and 
slowing  of  the  respiration.  The  nostrils  were  motionless, 
and  within  five  hours  complete  paralysis  of  the  muscles 
took  place.  The  reflex  excitability  gradually  diminished 
until  it  finally  disappeared.  An  orange-blossom  odor  was 
observed  about  the  frogs  which  were  poisoned  by  this 
ptomaine.  The  same  amount  of  ptomaine  injected  into  a 
greenfinch  produced  vomiting,  and  a  condition  of  weakness 
and  decreased  sensibility,  followed  soon,  however,  by  re- 
covery. A  rat  was  not  affected  by  0.020  'gramme  of  the 
free  base.     The  hydrochloride  acts  much  more  energetically. 

A  Base,  C10H15N,  was  isolated  by  O.  de  Coninck,  in 
1886  (Hugounenq,  page  21,  C.  Hendus,  1888),  from 
sea-polyps  in  an  advanced  stage  of  putrefaction,  together 
with  the  base  C8HnN.  The  method  employed  for  its  ex- 
traction was  that  of  Gautier  and  Etard  (see  page  164). 
It  forms  a  slightly  yellow,  viscous  liquid,  and  possesses  a 
pleasant  odor  resembling  that  of  blooming  broom.  Its 
density  is  about  1.18.    It  boils  at  about  230°  (uncorrected), 


chemistry  of  the  ptomaines.         203 

with  initial  decomposition.  In  water  it  is  but  slightly 
soluble,  readily  so  in  ether,  alcohol,  acetone,  and  ligroin. 
It  is  rapidly  oxidized  by  the  air,  becomes  brown,  and 
resinifies  but  does  not  absorb  carbonic  acid. 

The  Hydrochloride,  C10H15N.HC1,  forms  fine  yel- 
lowish, very  deliquescent  needles,  which  in  the  presence  of 
a  trace  of  air  are  at  once  colored  red  ;  if  more  air  is  present 
the  red  changes  to  a  brown,  and  in  the  open  air  a  resin  is 
formed  the  same  as  from  the  free  base.  It  is  very  easily 
soluble. 

The  Hydrobromide,  C10H15N.HBr,  crystallizes  in  a 
network  of  fine  deliquescent  needles,  which  become  likewise 
red  on  exposure  to  air.  It  is  very  soluble  in  water ;  less 
so  in  strong  alcohol,  and  almost  insoluble  in  ether. 

The  Platinochloride,  (C10H15N.HCl)2PtCl4,  forms  a 
dark -red  powder,  which  is  insoluble  in  cold  water ;  very 
soluble  in  warm  water.  It  can  be  kept  in  dry  air  ;  in  moist 
air  it  loses  hydrochloric  acid  and  becomes  partially  oxidized. 
Boiling  water  decomposes  it.  (C10H15N.Cl)2PtC]2  forms 
clear-brown  plates,  which  are  stable  in  moist  air,  and  melt 
at  206°.  It  is  insoluble  in  cold  water,  soluble  in  boiling 
water,  but  decomposes.  In  recrystallizing,  warm  previously 
boiled  water  should  be  used. 

The  Aurochloride,  C10H15jN\HC1. AuC13,  occurs  as  a 
light-yellow  precipitate ;  insoluble  in  cold  water,  soluble  in 
warm  water.  It  is  decomposed  by  boiling  water  ;  is  stable 
when  kept  in  a  moist  atmosphere. 

The  Iodomethylate,  C10H15N.CH3I,  in  warm  alcoholic 
solution  yields,  on  the  addition  of  strong  potash,  a  bright- 
red  color,  which  soon  becomes  brown,  and  in  about  an 
hour  the  solution  shows  a  greenish-blue  fluorescence.  This 
rapidity  of  change  is  due  to  the  extreme  oxidizability  of  the 
ptomaine. 

O.  de  Coninck  considers  this  base,  as  well  as  C8HnN, 
as  belonging  to  the  pyridine  and  not  to  the  hydropyridine 
series. 

A  Base,  C10H17N,  was  described  by  Griffiths  (1890) 
as  derived  from  cultures  on  peptone-agar  of  the  bacterium 


204  BACTERIAL    POISON'S. 

allii,  a  germ  obtained  from  putrid  onions.  The  base 
(hydrochloride?)  forms  colorless,  prismatic,  microscopic, 
very  deliquescent  needles,  which  are  soluble  in  warm  water, 
alcohol,  ether,  and  chloroform.  It  gives  on0  a  hawthorn-like 
odor,  especially  when  warmed.  With  phosphomolybdic  acid 
it  yields  a  white ;  with  iodine  in  potassium  iodide  and  with 
tannic  acid  a  chestnut-colored  precipitate.  Nessler's  solu- 
tion produces  a  yellow  chestnut-colored  precipitate.  Picric 
acid  throws  down  a  yellow  slightly  soluble  deposit.  The 
platinochloride,  (C10H17N.HCl)2PtCl4,  is  yellow,  crystalline, 
and  difficultly  soluble  in  cold  water  and  in  alcohol ;  soluble 
in  warm  water.  Gold  chloride  produces  a  thick  yellow 
precipitate  soluble  in  water.  Dilute  sulphuric  acid  pro- 
duces a  violet-red  color.  The  base  is  apparently  a  hydro- 
coridine. 

A  Base,  C32H31N,  was  obtained  by  Delezhstier  (1889) 
and  is  said  to  be  the  alkaloid,  isolated  in  1879  by  Brouar- 
del,  which  in  its  chemical  and  physiological  properties  was 
described  as  similar  to  veratrine.  It  forms  an  almost  color- 
less oily  fluid,  which  possesses  a  hawthorn-like  odor.  It 
is  very  readily  oxidizable  and  yields  the  veratrine-like  re- 
actions only  in  the  presence  of  air.  It  is  soluble  in  alcohol, 
ether,  toluene,  and  benzene;  and  forms  well-defined  salts 
which  are  very  deliquescent.  It  appears  to  be  an  amine, 
and  in  its  composition  differs  from  cevadine  by  9H20. 
Nothing  is  stated  in  regard  to  its  source  or  method  of 
preparation.  The  analytical  results  given — C  =  89.41, 
H  =  7.3,  1ST  =  3.03  —  correspond  more  to  the  formula 
C34H33N. 

Ethylidenediamine  (?),  C2H8N2. — This  base  was  con- 
sidered at  first  by  Brieger  to  be  identical  with  ethylene- 
diamine,  but  subsequent  comparison  showed  this  to  be  an 
error.  Thus,  the  former  is  poisonous  and  does  not  form 
a  gold  salt,  while  the  latter  is  not  poisonous  and  does  form 
a  rather  difficultly  soluble  gold  salt.  Again,  ethylene- 
diamine  forms  a  platinochloride  which  is  almost  insoluble 
in  hot  water,  whereas  the  platinum  double  salt  of  the  pto- 


CHEMISTRY    OF    THE    PTOMAINES.  205 

ma'ine  is  much  more  easily  soluble.  Brieger  is,  therefore, 
inclined  to  think  that  it  is  identical  with  ethylidenediamine, 
CH3.CH(NH2)2,  rather  than  Avith  ethylenediamine,  which 
has  the  structure,  CH2.NH2.CH2.NH2.  This  ptomaine 
was  obtained  by  Brieger,  in  1885  (I.,  44),  from  decom- 
posing haddock  (see  Gadinine). 

The  free  base  can  be  obtained,  without  decomposition,  on 
distilling  the  hydrochloride  with  sodium  hydrate. 

The  Hydrochloride,  C2H8N2.2HC1,  crystallizes  in 
long  glistening  needles  which  are  readily  soluble  in  water, 
insoluble  in  absolute  alcohol.  It  gives  no  combination  with 
gold  chloride.  For  its  behavior  to  alkaloidal  reagents  see 
Table  I. 

The  Peatinochloride,  C2H8N2.2HCl.PtCl4  (Pt  = 
41.49  per  cent.),  forms  small  yellow  plates  which  are 
moderately  difficultly  soluble  in  water.  It  can  be  readily 
recrystallized  from  hot  water. 

Physiological  Action. — Frogs  seem  to  be  less  suscepti- 
ble to  the  action  of  this  poison  than  mice  or  guinea-pigs. 
In  the  latter,  it  produces  a  short  time  after  injection  an 
abundant  periodic  flow  of  secretion  from  the  nose,  mouth, 
and  eyes.  The  pupils  dilate  and  the  eyeballs  project. 
Violent  dyspncea  then  comes  on  and  predominates  until  the 
death  of  the  animal,  which  does  not  take  place  for  twenty- 
four  hours  or  more.     The  heart  is  stopped  in  diastole. 

Trimethylenedi amine  ('?),  C3H1()N2 '(?),  is  a  toxic  base 
isolated  by  Brieger  (1887)  from  cultures  of  the  comma 
bacillus  on  beef-broth.  It  may  be  stated  here  that  from 
the  same  source,  cholera  cultures,  Kunz  (1888)  obtained  a 
base  which  he  considered  to  be  identical  with  spermine  or 
ethyleneimine  (see  next  chapter).  It  is  present,  however,  in 
exceedingly  minute  quantity,  and  occurs  in  the  mercuric 
chloride  precipitate,  from  which  it  is  obtained  by  the  fol- 
lowing method  :  The  precipitate  is  decomposed  by  hydrogen 
sulphide,  the  filtrate  evaporated  to  dryness,  and  the  residue 
taken  up  with  absolute  alcohol  and  precipitated  by  an 
alcoholic   solution    of    sodium    picrate.       The   precipitate 

10 


206  BACTERIAL    POISONS. 

thus  obtained  consists  of  the  picrates  of  cadaverine,  crea- 
tinine, and  of  this  new  base.  It  is  boiled  with  absolute 
alcohol  to  remove  the  insoluble  cadaverine  picrate ;  the 
filtrate  is  evaporated  to  expel  the  alcohol,  and  the  bases 
then  converted  into  the  platinum  double  salts,  whereby  the 
easily  soluble  creatinine  platinochloride  can  be  separated 
from  the  corresponding  less  soluble  compound  of  the  new 
base. 

Owing  to  the  small  quantity  of  this  substance  present,  a 
complete  study  of  its  properties  has  not  as  yet  been  made. 
It  gives  difficultly  soluble  precipitates  with  gold  chloride 
and  with  platinum  chloride ;  the  compound  with  the  latter 
crystallizes  in  long  needles.  With  picric  acid  it  gives  a 
precipitate  consisting  of  felted  needles,  which  resemble 
creatinine  picrate;  they  melt  at  198°.  Phosphomolybdic 
acid  yields  a  precipitate  crystallizing  in  plates,  while  potas- 
sium-bismuth iodide  gives  dark-colored  fine  needles.  From 
its  physiological  action  it  seems  to  be  identical  with  the 
basic  substance  isolated  from  choleraic  bodies  by  different 
observers.  It  causes  violent  convulsions  and  muscle 
tremor. 

Besides  trimethylenediamine  another  toxine  was  obtained 
by  Brieger  from  cholera  cultures,  but  in  quantity  insuffi- 
cient for  analysis.  It  was  obtained  from  the  mercuric 
chloride  filtrate  after  elimination  of  methy lamine,  trimethyl- 
amine,  and  traces  of  choline  and  creatinine,  as  an  insoluble 
platinum  double  salt.  Subcutaneous  injection  of  this  base 
into  mice  produced  a  paralysis-like  lethargic  condition, 
slowing  of  respiration  and  heart's  action,  lowering  of 
temperature,  and  finally,  death  in  twelve  to  twenty-four 
hours.     In  some  cases  bloody  stools  were  passed. 

Putrescine,  C4H12ISr2,  is  a  diamine  which  almost  in- 
variably occurs  together  with  cadaverine,  with  which  it  is 
apparently  closely  related.  This  base  was  also  discovered 
by  Brieger  in  1885  (II.,  42),  who  has  obtained  it  from 
putrefying  human  internal  organs  (for  four  months  at  a 
low  temperature  without  access  of  much  oxygen) ;  and 
from  the  same  material,  decomposing  at  the  ordinary  tern- 


CHEMISTKY    OF    THE    PTOMAINES.  207 

perature  of  the  room,  for  from  three  clays  to  three  weeks. 
It  has  also  been  obtained  from  herring,  twelve  days  in 
spring ;  from  pike,  six  days  in  summer ;  from  haddock, 
two  months  (Bocklisch).  Also  from  putrid  mussel,  six- 
teen days  (Brieger)  •  and  from  human  as  well  as  horse 
flesh.  Brieger  has  obtained  it  from  cultures  of  the  bac- 
teria of  human  feces  on  gelatin,  and  in  small  quantity  in 
rather  old  cultures  of  the  comma  bacillus  on  beef-broth  ;  in 
larger  quantity  in  cultures  of  the  same  germ  on  blood- 
serum. 

Udranszky  and  Baumann  in  1888  demonstrated  the 
existence  of  putrescine  and  cadaverine  in  the  urine  of  cyst- 
inuria,  the  former  constituting  about  one-third  of  the 
total  amount  of  the  two  bases  present.  In  the  feces  of  the 
same  patient,  on  the  contrary,  putrescine  constituted  by  far 
the  greater  quantity,  while  cadaverine  formed  but  10  to  15 
per  cent.  Normal  feces,  as  well  as  the  feces  of  various 
diseases  with  the  possible  exception  of  cholera  stools,  are 
free  from  diamines.  It  would  seem,  therefore,  that  these 
bases  occur  in  cystinuria  as  the  result  of  putrefactive 
changes  going  on  in  the  intestines ;  becoming  partly  ab- 
sorbed they  appear  in  the  urine.  In  two  cases  of  cystin- 
uria, reported  by  Brieger  and  Stadthagen,  cadaverine 
was  found  almost  solely  present  in  the  urine. 

According  to  Mester  the  diamines  are  proportionate  to 
the  amount  of  cystin  excreted,  and  therefore  constitute  a 
fixed  symptom,  the  cause  of  which  is  the  same  as  that  of 
the  cystinuria. 

Although  putrescine  is  recognizable  on  about  the  fourth 
day  of  the  putrefaction,  yet  it  does  not  occur  in  appreciable 
quantity  until  about  the  eleventh  day.  The  amount  that 
is  formed  increases  as  the  putrefaction  goes  on,  so  that  a 
considerable  quantity  may  be  obtained  after  two  or  three 
weeks.  A  very  good  source  for  the  preparation  of  putrescine, 
cadaverine,  and  neuridine  is  gelatin  which  has  been  allowed 
to  decompose  in  contact  with  water  for  some  weeks. 
Neuridine  is,  apparently,  formed  first,  but  is  soon  replaced 
by  the  former  two  bases.  In  the  process  of  extraction  it 
is  first  obtained  in  the  alcoholic  mercuric  chloride  precipi- 


208  •      BACTERIAL    POISONS. 

tate.  For  its  separation  from  cadaverine  and  other  accom- 
panying bases,  see  page  220. 

From  the  urine  of  cystinuria  it  is  best  obtained  by  pre- 
cipitation with  benzoyl  chloride  (Baumann's  method). 
For  this  purpose  about  1500  c.c.  of  urine  are  treated 
with  200  c.c.  of  sodium  hydrate  solution  (10  per  cent.), 
then  20  to  25  c.c.  of  benzoyl  chloride  is  added,  and  the 
whole  shaken  till  the  odor  of  the  latter  disappears.  The 
yellowish-white  precipitate  which  forms  may  consist  of  in- 
soluble phosphates,  carbohydrates,  polyatomic  alcohols,  and 
diamines.  The  cyst  in  compound  is  precipitated  only  in 
concentrated  solutions.  The  precipitate  contains  from  a 
half  to  two-thirds  of  the  diamines  present ;  it  is  filtered 
off,  digested  with  warm  alcohol,  and  the  solution  filtered. 
The  alcoholic  filtrate  is  concentrated  and  then  poured  into 
about  thirty  times  its  volume  of  cold  water.  The  diamine 
compounds  then  crystallize  out.  To  separate  the  two  dia- 
mines they  are  redissolved  in  just  sufficient  warm  alcohol 
to  effect  solution,  and  this  is  then  poured  into  about  twenty 
times  this  volume  of  ether.  The  putrescine  benzoyl  com- 
pound is  thus  thrown  out  of  solution.  The  filtrate  from 
this  on  concentration  yields  the  cadaverine  compound.  To 
isolate  that  portion  of  the  diamines  which  remained  in  the 
original  filtrate  with  benzoyl  cystin,  it  is  acidulated  with  sul- 
phuric acid  and  extracted  with  ether.  The  residue  obtained 
on  evaporating  the  ethereal  solution  is  first  neutralized  with 
a  12  per  cent,  sodium  hydrate  solution,  then  mixed  with 
three  to  four  times  its  volume  of  the  same  solution.  The 
precipitate  which  forms  consists  of  the  sodium  compounds 
of  benzoyl  cystin  and  the  diamines.  It  is  washed  with 
sodium  hydrate,  and  the  two  compounds  separated  by  their 
different  solubilities  in  water — the  cystin  compound  is 
readily  soluble,  that  of  the  diamines  insoluble.  To  purify 
the  benzoyl  diamines  they  are  dissolved  in  warm  alcohol 
and  precipitated  with  excess  of  water. 

Putrescine  (from  putresco,  to  rot,  to  putrefy)  is  a  water- 
clear,  rather  thin  liquid  which  fumes  in  the  air  and  has  a 
a  peculiar  semen-like  odor,  almost  undistinguishable  from 
that  of  cadaverine,  and  reminding  one  somewhat  of  the 


CHEMISTKY    OF    THE    PTOMAINES.  209 

pyridine  bases.  It  absorbs  carbonic  acid  energetically  from 
the  air,  without  losing  thereby  the  repulsive  odor.  The 
boiling-point  of  the  free  base,  as  ordinarily  obtained,  is 
about  135°.  It  is  not  decomposed  by  distillation  with 
potassium  hydrate,  and  is  rather  difficultly  volatile  with 
steam.  With  acids  it  forms  beautiful  crystalline  salts. 
Putrescine  unites  with  water,  like  ethylenediamine,  to  form 
a  hydrate,  and  this  water  can  only  be  removed  by  distilla- 
tion with  metallic  sodium.  The  perfectly  anhydrous  base 
boils  at  156°-157°,  and  then  solidifies  to  plates  (Briegeu), 
which  melt  at  24°  (Udranszky  and  Baumann).  The 
synthetic  base  boils  at  158°-160°,  and  melts  at  23°- 
24°  (Ladenburg).  Like  cadaverine  it  is  difficultly  solu- 
ble in  ether. 

The  constitution  of  putrescine  has  been  determined  by 
Udranszky  and  Baumann  (1888).  They  showed  that 
the  dibenzoyl  compound  of  putrescine  was  identical  with 
that  of  the  synthetic  tetramethylenediamine  and  of  the 
base  which  they  found  in  the  urine  of  cystinuria. 

Putrescine,  therefore,  is  tetramethylenediamine,  a  homo- 
logue  of  cadaverine,  and  its  rational  formula  is  : 

NH2.CH2.CH2.CH2  CH2.NH2. 

The  same  authors  (Zeitschr.  f.  Physiol.  Chem.  13,  591) 
point  out  that  diamines  may  possibly  occur  in  putrefaction 
as  the  result  of  oxidation  of  monamines.  Thus,  putrescine 
might  arise  from  methylamine  according  to  the  equation  : 

CH3.CH2.NH2  CH  —  CH  —  NH2 

+  0=|  +  ELO. 

CH3.CH2.NH2  CH— CH— NH2 

In  a  similar  manner  cadaverine  might  form  from  ethyl 
and  propylamine. 

Putrescine  can  be  prepared  synthetically,  according  to 
Ladenburg's  method,  by  converting  ethylene  bromide  into 
the  cyanide  and  then  reducing  this  by  means  of  sodium 
in  absolute  alcohol. 

On  heating  the  concentrated  aqueous  solution  of  the 
hydrochloride  with  potassium  nitrite  there  is  produced  an 


210  BACTERIAL    POISONS. 

oil,  soluble  in  water,  from  which  it  can  be  extracted  with 
ether.  This  oil,  on  treatment  with  phenol  and  sulphuric 
acid,  gives  Liebermann's  nitroso-reaction,  which  would 
seem  to  show  that  putrescine  is  not  a  primary  diamine 
(butylenediamine),  but  is  rather  a  secondary  diamine 
(Brieger,  II.,  42).  As  a  primary  diamine  it  should  take 
up,  on  repeated  treatment  with  methyl  iodide,  six  methyl 
radicals ;  whereas,  if  it  is  a  secondary  diamine,  only  four 
methyl  radicals  can  enter  the  molecule.  Thus,  to  illustrate, 
methylamine,  CH3.NH2  (a  primary  amine),  combines  with 
three  molecules  of  methyl  iodide  to  form  (CHg^N.HI. 
Similarly,  dimethylamine  (CII3)2.NH,  requires  only  two 
molecules  to  form  (CH3)4N.HI.  In  the  case  of  diamines, 
double  this  number  of  methyl  groups  is  required  to  effect 
complete  saturation.  As  a  matter  of  fact,  Brieger  (III , 
101),  on  treating  putrescine  with  methyl  iodide,  has  suc- 
ceeded in  introducing  four,  and  only  four,  methyl  radicals. 
From  this,  however,  it  does  not  follow  that  putrescine  is 
not  a  primary  amine,  since  cadaverine,  an  unquestioned 
primary  diamine,  yields  a  substitution  compound  contain- 
ing only  two  methyl  groups  (see  p.  215). 

The  tetra-methyl  substitution-product  of  putrescine, 
C4H8(CH3)4N2,  can  be  distilled  without  decomposition.  The 
free  base  crystallizes  in  long  prisms.  The  hydrochloride 
forms  small  needles  which  are  easily  soluble ;  with  phos- 
photungstic  acid  it  gives  a  white  crystalline  precipitate, 
with  phosphomolybdic  acid  a  yellow  crystalline  precipitate, 
with  picric  acid  needles.  Potassium-bismuth  iodide  gives 
a  brownish-red  amorphous  deposit,  while  the  potassium 
mercuric  iodide  forms  prisms.  Gold  chloride  yields  diffi- 
cultly, and  platinum  chloride  easily  soluble  octahedra ; 
aqueous  mercuric  chloride  forms  needles. 

The  aurochloride  has  the  formula  C8H22lSr2.2AuCl4. 

This  tetra-methyl  derivative  of  putrescine  is  enormously 
poisonous  as  compared  with  putrescine.  The  symptoms 
are  the  same  as  those  produced  by  muscarine  or  neurine. 
They  are  :  abundant  salivation ;  dyspnoea — respiration  at 
first  increases,  then  decreases ;  contraction  of  the  pupils  ; 
paralysis^ of  the  muscles  of  the  limbs  and  trunk ;   increased 


CHEMISTRY    OF    THE    PTOMAINES.  211 

peristaltic  action  of  the  intestines,  ejaculation  of  semen, 
dribbling  of  urine,  and,  finally,  violent  clonic  convulsions. 
In  the  case  of  mice  and  guinea-pigs  the  convulsions  are 
prominent  immediately  after  the  injection  of  the  poison. 

Putrescine  Hydrochloride,  C4H12N2.2HC1,  forms 
long  colorless  needles,  which  are  very  easily  soluble  in 
water  ;  difficultly  so  in  dilute  alcohol ;  entirely  insoluble  in 
absolute  alcohol,  and  can  thus  be  separated  from  cadav- 
erine  hydrochloride.  To  accomplish  this  separation  it  is, 
perhaps,  better  to  dissolve  the  mixture  of  the  hydrochlo- 
rides in  hot  96  per  cent,  alcohol.  On  cooling  the  solution 
thus  obtained,  the  putrescine  salt  crystallizes  out,  whereas 
that  of  cadaverine  remains  in  solution.  Putrescine  hydro- 
chloride differs  from  cadaverine  hydrochloride  in  that  it  is 
not  hygroscopic  and  can  be  exposed  for  days  to  the  air 
without  suffering  any  change  on  the  surface  of  the  crystals. 

For  the  behavior  of  the  free  base  and  the  hydrochloride 
to  alkaloidal  reagents,  see  Table  I.  Putrescine  is  not  toxic, 
though  it  possesses  some  marked  physiological  properties 
(see  Cadaverine,  page  215).  According  to  ScHEURLEisr 
putrescine,  like  cadaverine,  produces  inflammation,  suppu- 
ration, and  necrosis.  It  is  not  poisonous  to  dogs  (Udran- 
szky  and  Baumann).     It  is  optically  inactive. 

The  Peatinochloride,  C4H12N2.2HCl.PtCl4  (Pt  = 
39.16  per  cent.),  often  appears  under  the  microscope  in  the 
form  of  cholesterine-like  plates.  In  the  pure  condition  it 
appears  as  six-sided  plates,  which  are  superposed  in  layers. 
The  crystals  possess  a  splendid  silvery  lustre,  and  are  rather 
difficultly  soluble  in  cold  water  ;  less  so  in  hot  water. 

The  Aurochloride,  C4H12N2.2HC1.2AuCl3  +  2H20, 
crystallizes  likewise  in  plates,  which  are  difficultly  soluble 
in  cold  water.  It  can,  therefore,  be  readily  separated  from 
cadaverine  aurochloride,  which  is  easily  soluble  in  water. 
The  water  of  crystallization  can  be  driven  off  completely 
only  at  110°  (Brieger).  According  to  Bocklisch,  it 
loses  this  water  on  standing  over  sulphuric  acid,  or  on 
heating  at  100°. 

The  Picrate,  C4H12N2.2C6H2(N02)3OH,  is  difficultly 
soluble,  and  crystallizes  from  a  hot  aqueous   solution  in 


212  BACTERIAL    POISONS. 

needles ;  from  hot  aqueous  alcohol,  on  cooling,  in  yellow 
plates.  It  begins  to  brown  at  230°,  and  on  further  heating 
becomes  darker,  till  finally,  at  250°,  it  decomposes  with 
rapid  evolution  of  gas  (Bocklisch). 

The  Carbonate  is  crystalline. 

The  Mercury  double  salt  is  easily  soluble  in  a  large 
quantity  of  water,  and  can  thus  be  separated  from  the 
cadaverine  salt,  which  is  difficultly  soluble.  From  hot  con- 
centrated aqueous  solution  it  crystallizes  in  needles. 

The  Dibenzoyl  -  putrescine,  C4H8(NHCOC6H5)2, 
forms  silky  plates  or  long  needles,  which  are  more  diffi- 
cultly soluble  in  hot  alcohol  than  those  of  the  cadaverine 
compound.  From  this  solution  it  is  reprecipitated  by  ad- 
dition of  water  or  ether.  Its  melting-point  is  175°.  It 
sublimes  without  decomposition. 

Cadaverine,  C5H14N2,  is  a  diamine  isomeric  with  sap- 
rine  and  neuridine,  and,  like  the  latter,  it  occurs  very  fre- 
quently in  decomposing  animal  tissues.  Twelve  isomers  of 
this  composition  are  possible.  Another  isomer,  gerontine  (see 
next  chapter)  has  been  described  by  Grandis  (1890).  It  is 
a  very  striking  fact,  that  in  ordinary  putrefaction  as  choline 
disappears  the  diamines  appear  and  increase  in  quantity 
according  as  the  time  of  putrefaction  is  extended.  It  is 
also  worthy  of  note  that  cadaverine  appears  in  putrefaction 
before  putrescine.  It  has  been  obtained  by  Brieger  (1885) 
from  human  lungs,  hearts,  livers,  ete.  (hence  the  name), 
which  were  allowed  to  putrefy  at  the  ordinary  temperature 
for  three  days  ;  from  the  same  organs,  and  from  horseflesh, 
after  four  months  in  a  closed  vessel  at  — 9°  to  +  5°  ; 
from  putrid  mussel  after  sixteen  days  ;  from  putrid  egg  and 
blood  albumin.  It  seems  to  be  a  constant  product  of  the 
growth  of  the  comma  bacillus,  irrespective  of  the  soil  on 
which  it  is  cultivated. 

Bocklisch  has  isolated  it  from  perch  and  pike,  six  days 
in  midsummer ;  from  herring,  twelve  days  in  spring ;  from 
haddock,  two  months  at  a  low  temperature ;  from  cultiva- 
tions of  Finkler  and  Prior's  vibrio  proteus  on  beef- 
broth,  thirty  to  thirty-five  days  at  37°  to  38°  (Ber.  20, 


CHEMISTRY    OF    THE    PTOMAINES.  213 

1441).  Cadaverine  seems  to  be  a  constant  product  of  the 
activity  of  the  genus  vibrio,  inasmuch  as  it  does  not  occur 
in  cultures  in  which  this  genus  is  absent.  Thus,  it  is  not 
present  in  the  excrements  of  healthy  or  typhoid  patients  ;  in 
cultures  of  Emmerich's  bacillus,  of  Eberth's  bacillus,  and 
of  the  pyogenic  bacteria.  It  is  said  to  occur  in  cultures  of 
the  bacillus  of  hog-cholera  (v.  Schweinitz).  Oechsner 
de  Coninck  has  found  it  in  putrid  jelly-fish  (Hugounenq, 
page  23).  It  is  present  with  putrescine  in  the  urine  and 
feces  of  cystinuria  (Udranszky  and  Baumann,  1888). 
The  odor  of  cholera  stools  and  the  breath  of  cholera  patients 
may  be  possibly  due  to  cadaverine,  although  the  base  has 
not  been  demonstrated  in  such  cases.  It  has  also  been  ob- 
tained from  caviar. 

Cadaverine  occurs  in  the  mercuric  chloride  precipitate, 
from  which  it  is  isolated  according  to  the  methods  given  on 
pages  206  and  221.  For  its  isolation  and  separation  from 
jratrescine  by  the  use  of  benzoyl  chloride,  see  page  208. 

This  base  was  at  first  ascribed  the  formula  C5H16N2,  but 
subsequent  researches  led  Brieger  and  Bocklisch  to  the 
adoption  of  the  formula  C5HI4N2.  In  1883,  Ladenburg 
prepared,  as  the  first  step  in  the  synthesis  of  piperidine,  a 
base,  pentamethylenediamine,  possessing  the  same  empirical 
formula  as  cadaverine,  and  later  (Ber.  18,  2956)  he  showed 
the  possibility  of  the  identity  of  these  two  bases.  This  led 
to  their  direct  comparison  and  the  successful  establishment 
of  their  identity.  In  fact,  Ladenburg,  as  a  crucial  test  of 
the  identity,  converted  cadaverine  into  piperidine,  and  found 
the  latter  base  to  agree  entirely  in  its  chemical  and  physical 
properties  with  those  of  the  natural  alkaloid  (Ber.  19,  2586). 
Ladenburg,  however,  observed  one  apparent  difference 
between  cadaverine  and  pentamethylenediamine,  and  that 
was  in  the  composition  of  the  mercury  double  salts.  That 
of  the  former  base,  whether  obtained  from  alcoholic  or 
aqueous  solution  (Bocklisch,  Ber.  20,  1441),  was  found 
to  combine  with  four  molecules  of  mercuric  chloride ; 
whereas  the  double  salt  of  pentamethylenediamine  was 
found  by  Ladenburg  to  contain  only  three  molecules  of 
mercuric  chloride.      Subsequently  he  found  that  he   had 

10* 


214  BACTERIAL    POISONS. 

prepared  this  salt  by  mixing  the  aqueous  solutions  of  the 
hydrochloride  of  the  base  and  of  the  mercuric  chloride  in 
the  molecular  ratio  of  1  to  4,  and  on  using  a  larger  excess 
of  mercuric  chloride  he  obtained  a  salt  containing  four 
molecules  of  mercuric  chloride  (Ber.  20,  2216).  The  com- 
plete identity  of  these  two  bases  has,  therefore,  been  estab- 
lished.    The  constitutional  formula  of  cadaverine  is,  there- 

NH—  CH2— CH2-CH—  CH2— CH—  NII2. 

Cadaverine  can  be  prepared  synthetically  according  to 
Ladenburg's  method.  For  this  purpose  trimethylene 
bromide  is  converted  into  the  cyanide,  and  this  is  then 
reduced  by  sodium  in  absolute  alcohol. 

Cadaverine  forms  a  somewhat  thick,  water-clear,  syrupy 
liquid,  which  possesses  an  exceedingly  unpleasant  odor, 
resembling  somewhat  that  of  coniine  (piperidine)  and  of 
samen.  When  dehydrated  with  potassium  hydrate  it  boils 
at  115°-120°  (Brieger).  It  boils  at  175°  (Brieger,  III., 
98),  and  fumes  in  the  air.  The  base  eagerly  absorbs  car- 
bonic acid  from  the  air,  and  solidifies  into  a  crystalline 
mass,  the  carbonate.  It  is  volatile  with  steam,  and  can  be 
distilled,  without  decomposition,  even  in  presence  of  sodium 
or  barium  hydrate,  or  soda  lime.  JNTeuridine,  its  isomer, 
decomposes  under  these  circumstances.  When  heated  with 
alcoholic  potash  and  chloroform  it  does  not  give  the  iso- 
nitril  reaction,  nor  does  it  give  the  characteristic  odor  of  oil 
of  mustard  on  treatment  with  carbon  disulphide  and  mer- 
curic chloride.  The  absence  of  these  reactions  at  first 
induced  Brieger  to  conclude  that  cadaverine  and  putres- 
cine  were  not  primary  amines,  but  Ladenburg  (1885) 
showed  that  this  conclusion  was  not  justifiable.  These  two 
reactions  are  given  by  primary  monamines,  but  in  this  case 
they  are  not  given  by  cadaverine,  a  primary  diamine.  It 
is  probable  that  this  behavior  holds  true  for  all  diamines. 

Cadaverine  is,  undoubtedly,  identical  with  the  so-called 
"animal  coniine,"  which  has  been  isolated  at  various  times 
from  cadavers. 

Cadaverine   and  putrescine  were    at  first   regarded   as 


CHEMISTRY    OF    THE    PTOMAINES.  215 

physiologically  indifferent,  but  more  recent  investigations 
by  Scheurlen,  Grawitz,  and  others,  show  that  both  these 
bases  are  capable  of  producing  strong  inflammation  and 
necrosis.  According  to  Behrtng,  in  large  doses  it  is 
poisonous  to  mice,  rabbits,  and  guinea-pigs ;  it  is  not 
poisonous  to  dogs  (Udranszky  and  Baumann).  Cadav- 
erine  is  one  of  those  substances  which  can  set  up  suppura- 
tion in  the  absence  of  bacteria.  In  cholera  Asiatica  the 
necrosis  of  the  intestinal  epithelium  is  quite  common,  and  it 
would  seem  that  this  pathological  change,  as  well  as 
the  muscular  spasms  and  algidity,  are  due  to  the  pres- 
ence of  these  bases.  It  should  be  noted,  however,  that 
Udranszky  and  Baumann  failed  to  obtain  any  sign  of 
intestinal  irritation  on  feeding  dogs  enormous  doses  of 
cadaverine.  Besides  these  local  effects,  they  prevent,  even 
in  small  quantity,  the  coagulation  of  blood,  and  render  it 
"  laky."  According  to  Grawitz,  cadaverine  seems  to 
hinder  the  growth  of  bacteria.  The  cystitis  observed  in 
cystinuria  may  possibly  be  clue  to  the  presence  of  cadaverine 
and  putrescine  in  the  urine.  Both  bases  are  optically 
inactive. 

When  cadaverine  is  treated  with  methyl  iodide,  a  base 
is  obtained,  the  hydrochloride  of  which  gives  with  pla- 
tinum chloride  a  double  salt,  having  the  composition : 
C5H12(CH3)2>T2.2HCl.PtCl4.  This  new  base,  therefore,  is 
cadaverine  in  which  two  atoms  of  hydrogen  have  been 
replaced  by  two  methyl  radicals.  The  platinochloride  of 
this  derivative  forms  long,  clear  red  needles,  which,  unlike 
those  of  cadaverine,  do  not  change  their  shape  on  repeated 
recrystallization.  It  is  moderately  difficultly  soluble  in 
water  (Brieger,  II.,  41).  Since  cadaverine  is  a  primary 
diamine  it  should  combine  with  six  molecules  of  methyl 
iodide  to  form  a  saturated  compound.  This,  however,  has 
not  been  obtained. 

The  Hydrochloride,  C5H14N2.2HC1,  crystallizes  in 
beautiful,  long  deliquescent  needles  (Brieoer).  According 
to  Bocklisch,  it  forms  long,  colorless  needles  or  prisms ; 
crystallizes  from  alcohol  in  plates,  and  is  not  deliquescent 
except  on  long  standing.     It  is  soluble  in  water,  alcohol, 


216  BACTERIAL    POISONS. 

alcohol-ether ;  but  is  insoluble  in  absolute  alcohol,  ether, 
etc.  It  can  readily  be  separated  from  putrescine  hydro- 
chloride by  its  solubility  in  96  per  cent,  alcohol  (Bock- 
lisch).  The  strictly  pure  base,  as  well  as  the  hydro- 
chloride, does  not  give  a  blue  color  with  ferric  chloride  and 
potassium  ferricyanide.  For  reactions  of  the  hydrochloride 
and  of  the  free  base,  see  Table  I. 

Cadaverine  hydrochloride  on  dry  distillation  decomposes 
into  NH3,  HC1,  and  piperidine,  C5HulNr.  The  latter  is  a 
well-known  poisonous  alkaloid  which  exists  in  the  combined 
state  in  black  pepper.  It  is  not  known  whether  this  change, 
whereby  the  non-poisonous  cadaverine  is  converted  into  a 
toxic  base,  can  take  place  under  the  influence  of  bacteria 
during  the  processes  of  putrefaction  or  not.  However,  it 
does  not  seem  improbable  that  this  simple  chemical  change 
should  be  effected  through  the  action  of  living  organisms  ; 
for  Schmidt  has  already  shown  that  the  almost  physiologi- 
cally indifferent  choline,  when  subjected  to  the  action  of 
the  bacteria  of  hay-infnsion,  decomposes  into  a  neurine-like 
base  possessing  a  muscarine-like  action,  and  under  certain 
conditions  it  yields  a  base  which  in  its  action  resembles 
pilocarpine. 

The  Sulphate  likewise  forms  beautiful,  well-formed 
needles,  and  in  its  solubility  corresponds  to  the  hydro- 
chloride. 

The  Platinochloride,  C5H14N2.2HCl.PtCl4  (Pt  = 
38.08  per  cent.),  crystallizes  after  some  time,  on  the  addition 
of  platinum  chloride  to  a  not  too  concentrated  solution  of  the 
hydrochloride,  in  the  form  of  long,  beautiful  orange-red 
needles  (Bocklisch).  Ordinarily  it  is  obtained  at  first  in 
long,  dirty  red  needles,  which  on  repeated  recrystallization 
becomefclearer  and  assume  a  form  similar  to  that  of  ammo- 
nium  platinochloride.  It  forms  chrome-yellow  rhombic 
prisms  which  are  short  and  octahedra-like.  In  polarized 
light  they  are  strongly  double  refracting.  It  is  very  slightly 
soluble  in  cold  water ;  can  be  recrystallized  from  hot  water 
(Bocklisch).  Its  solubility  in  water  at  12°  is  1  to  113- 
114.    lit  decomposes  at  235°-236°. 

TheAuROCHLORiDE,C5HuN2.2HC1.2AuCl3(Au=50.41 


OHEMISTEY    OF    THE    PTOMAINES.  217 

per  cent.),  crystallizes  partly  in  cubes,  and  partly  in  long 
needles  which  at  first  possess  a  bright  lustre,  but  under  the 
desiccator  soon  effloresce  and  become  opaque.  The  water 
of  crystallization  is  completely  removed  on  standing  over 
sulphuric  acid.  It  is  very  easily  soluble,  and  melts  at  188° 
(Bocklisch). 

The  Picrate,  C5H14N2.2C6H2(N02)3OH,  forms  yellow 
plates  which  are  difficultly  soluble  in  cold  water.  From 
hot  water  it  crystallizes  in  long  prisms,  which  melt  at  221° 
with  decomposition.  It  is  insoluble  in  absolute  alcohol 
and  can  be  recrystallized  from  hot  dilute  alcohol. 

Cadaverine  hydrochloride  combines  with  mercuric  chlo- 
ride, when  the  aqueous  solutions  of  these  two  salts  are 
mixed  in  the  molecular  ratio  of  1  to  4,  to  form  C5H14ISr2. 
2HC1.3HgCl2.  This  salt  can  be  recrystallized  from  hot 
water  (Ladenburg).  When  an  excess  of  mercuric  chlo- 
ride is  used  the  double  salt  has  the  composition  C5H14N2. 
2HC1.4HgCl2.  This  last  salt  melts  at  216°  (Ladenburg)  ; 
at  214°  (Bocklisch).  It  is  difficultly  soluble  in  cold 
water ;  from  hot  water  it  crystallizes  in  needles  or  plates 
(Bocklisch). 

The  Neutral  Oxalate,  C5H14N2.H2C204+2H20,  was 
prepared  by  Bocklisch  by  adcting  a  little  less  than  the  cal- 
culated quantity  of  alcoholic  oxalic  acid  to  the  cadaverine. 
The  precipitate  may  be  recrystallized  from  hot  dilute  alco- 
hol, when  it  is  obtained  in  the  form  of  needles,  which  melt 
at  about  160°  and  at  the  same  time  give  off  gas. 

The  Acid  Oxalate,  C5H14N2.2H2C204+H20,  is  made 
by  bringing  the  neutral  salt  into  alcoholic  oxalic  acid.  It 
is  soluble  in  hot  dilute  alcohol,  and  recrystallizes  from  it  in 
quadratic  plates,  sometimes  in  glistening  needles.  It  melts 
at  143°  with  decomposition.  After  it  has  been  dried 
over  sulphuric  acid,  it  loses,  on  being  heated  to  105°-110°, 
one  molecule  of  water  (Bocklisch,  Ber.  20,  1441).  The 
insolubility  of  these  oxalates  in  absolute  alcohol  shows  the 
fallacy  of  Tamba's  distinction  between  ptomaines  and  vege- 
table alkaloids.     (See  page  186.) 

The  dibenzoyl  derivative,  CSH10(NHCOC6H5)2,  crys- 
tallizes  in   long    needles   and   plates,    readily   soluble  in 


218  BACTERIAL    P0I30XS. 

alcohol,  difficultly  so  in  ether,  and  insoluble  in  water  ; 
hence  the  alcoholic  solution  can  be  precipitated  by  addition 
of  water  or  ether  (separation  from  the  putrescine  compound, 
see  p.  208).  It  melts  at  129°-130°.  It  is  not  changed  by 
boiling  with  dilute  acids  and  alkalies ;  but  boiling  with 
concentrated  hydrochloric  or  sulphuric  acids  for  a  long 
time  finally  breaks  it  up. 

Xe  uridine,  C-HUX2,  was  the  first  diamine  isolated  from 
animal  tissues  f  Brieger,  1883).  It  is  one  of  the  most 
common  products  of  putrefaction,  and  as  such  has  been 
obtained  by  Brieger  from  putrid  horseflesh,  beef,  human 
muscle,  five  to  six  days ;  from  haddock,  five  days  in  sum- 
mer ;  from  cheese,  six  weeks  in  summer ;  from  gelatin, 
ten  days  at  35°  ;  from  decomposing  human  internal  organs, 
three  to  eleven  days  ;  from  cultures  of  the  Eberth  bacillus, 
with  my  dine. 

Bocklisch  lias  obtained  it  from  perch,  six  days  in 
summer ;  from  barbel  after  three  days  in  summer. 

It  has  also  been  obtained  from  fresh  eggs  in  the  prepa- 
ration of  choline  by  heating  with  baryta ;  and  also  from 
fresh  brain  by  heating  with  2  per  cent,  hydrochloric  acid 
(Brieger,  I.,  57-61).  Ehrenbeeg  (1887)  found  it  in 
poisonous  sausage  and  obtained  it  by  growing  a  bacillus 
from  this  source  on  liver  and  meat  bouillon. 

Xeuridine  is  almost  invariably  accompanied  by  choline, 
and  as  the  duration  of  putrefaction  increases,  the  latter 
gradually  decreases  in  amount  and  yields  a  corresponding 
increase  in  trimethylamine,  whereas  the  yield  of  neuridine 
increases  from  day  to  day.  The  amount  of  neuridine 
formed  depends  upon  the  nature  of  the  organ  employed  in 
putrefaction.  The  greatest  yield  is  obtained  from  gelatin- 
ous tissues,  such  as  intestines ;  and  especially  from  pure 
gelatin.  On  the  other  hand,  such  tissues  as  the  spleen 
and  liver  yield  but  little. 

Xeuridine  comes  down  in  the  mercuric  chloride  precipi- 
tate (sometimes  it  occurs  in  the  filtrate),  and  can  then  be 
isolated  from  the  other  bases  present  in  a  number  of  ways. 
One  method  is  given  under  Gadinine.    Another  convenient 


CHEMISTRY    OF    THE    PTOMAINES.  219 

method  of  separation  is  to  precipitate  it  from  alcoholic 
solution  by  alcoholic  picric  acid.  The  picrate  thus  ob- 
tained is,  for  the  purpose  of  further  purification,  recrystal- 
lized  from  absolute  alcohol,  then  decomposed  by  extracting 
its  acid  solution  with  ether  (to  remove  the  picric  acid)  and 
evaporating  the  aqueous  solution  to  dryness.  The  residue 
is  now  extracted  with  alcohol  and  the  alcoholic  solution 
precipitated  by  alcoholic  platinum  chloride.  The  platino- 
chloride  can  now  be  recrystallized  from  hot  water. 

The  free  base,  as  obtained  by  the  treatment  of  the 
hydrochloride  with  moist  freshly  precipitated  silver  oxide, 
possesses  an  extremely  repulsive  odor,  similar  to  that  of 
human  semen.  On  evaporation  of  its  aqueous  solution  it 
yields  a  gelatinous-like  mass,  and  at  the  same  time  slowly 
decomposes.  It  does  not  crystallize  when  evaporated  in  a 
vacuum,  and  decomposes  even  under  these  conditions.  The 
same  disagreeable  odor  is  obtained  when  the  hydrochloride 
is  warmed  with  potassium  hydrate.  Brieg-er  (I.,  24)  re- 
gards this  decomposition-product  of  neuridine  as  au  oxida- 
tion product  of  the  original  substance. 

The  free  base  is  very  readily  soluble  in  water,  but  is 
insoluble  in  ether  and  absolute  alcohol ;  difficultly  soluble 
in  amyl  alcohol.  It  gives  white  precipitates  with  mercuric 
chloride,  neutral  and  basic  lead  acetates.  When  distilled 
with  fixed  alkali  it  yields  di-  and  tri-methylamine,  thus 
probably  showing  some  relation  to  neurine,  hence  the  name 
neuridine.  It  does  not  give  Hofmanx's  iso-nitril  reac- 
tion, but  it  does  not  follow  from  this,  as  shown  under 
cadaverine,  that  it  may  not  be  a  primary  diamine.  It  is 
isomeric  with  cadaverine,  saprine  and  gerontine. 

The  Hydrochloride,  C5H14lSr2.2HCl,  crystallizes  in 
long  needles  which  are  extremely  soluble  in  water  and 
in  dilute  alcohol,  but  are  insoluble  in  absolute  alcohol, 
ether,  benzol,  chloroform,  petroleum  ether,  benzine,  amyl 
alcohol,  etc.  Its  insolubility  in  absolute  alcohol  may  be 
used  to  effect  a  separation  from  choline  hydrochloride.  It 
can  be  recrystallized  from  slightly  warm  dilute  alcohol. 
Although  the  pure  salt  is  insoluble  in  the  reagents  just 
given,  nevertheless,  in  the  presence  of  other  animal  matter 


220  BACTERIAL    POISONS. 

it  is  dissolved  in  greater  or  less  quantity,  and  hence  can  be 
obtained  by  the  Stas-Otto  as  well  as  by  the  Dragen- 
dorff  method.  The  crystals  resemble  urea  in  form.  On 
heating  very  cautiously  the  salt  sublimes,  and  at  the  same 
time  appears  to  undergo  a  partial  internal  decomposition, 
inasmuch  as  many  of  the  groups  of  needles  in  the  sublimate 
are  colored  red  or  blue.  For  the  behavior  of  the  hydro- 
chloride with  the  alkaloidal  reagents,  see  Table  I. 

Pure  neuricline  is  not  poisonous,  but  as  long  as  it  is 
contaminated  with  other  putrefaction  products  it  possesses  a 
toxic  action  similar  to  that  of  peptotoxine.  This  holds 
true  for  the  other  non-poisonous  bases. 

The  Platinochloride,  C5H14N2.2HCl.PtCl4,  crystal- 
lizes in  beautiful  flat  needles.  Recrystallized  from  hot 
water,  it  forms  aggregations  of  small,  clear,  yellow  needles. 
It  is  readily  soluble  in  water,  from  which  it  is  precipitated 
on  the  addition  of  alcohol. 

The  Aurochloride,  C5H14N2.2HC1.2AuCl3,  is  rather 
difficultly  soluble  in  cold  water  (Bocklisch),  and  crystal- 
lizes on  cooling  of  the  hot,  saturated  solution  in  bunches  of 
clear,  yellow,  short  needles. 

The  Picrate,  C5H14N2.2C^H2(N02)3OH,  can  be  recrys- 
tallized  from  boiling  water,  in  which  it  is  very  difficultly 
soluble,  in  the  form  of  needles  united  in  plumose  groups. 
It  is  almost  insoluble  in  cold  water  ;  less  difficultly  soluble 
in  alcohol.  It  is  not  fusible,  but  begins  to  brown  and 
give  off  yellow  vapors  at  230°,  and  carbonizes  completelv 
at  250°. 

Saprine,  C5H14N2,  was  found  in  human  livers  and 
spleens  after  three  weeks'  putrefaction  (Brieger,  II.,  30, 
46,  58).  It  occurs  together  with  cadaverine,  putrescine, 
and  mydaleine  in  the  mercuric  chloride  precipitate.  To 
separate  these  bases,  Brieger  (1885)  used  the  following 
process  :  The  mercury  salts  were  decomposed  with  hydrogen 
sulphide,  the  filtrate  evaporated  to  dryness,  and  the  residue 
then  extracted  with  alcohol.  The  putrescine  hydrochloride 
is  insoluble  in  alcohol,  and  is  thus  removed.  The  alcoholic 
solution  was  treated  with  platinum  chloride,  which  precipi- 


CHEMISTRY    OF    THE    PTOMAINES.  221 

tated  the  greater  part  of  the  cadaverine.  The  mother- 
liquor,  on  concentration,  yielded  a  mixture  of  the  platino- 
chlorides  of  cadaverine  and  saprine.  Each  successive  crop 
contained  more  of  the  -saprine  double  salt.  The  two  kinds 
of  crystals  were  now  separated  by  means  of  a  magnifying- 
glass.  The  saprine  platinochloride  thus  obtained  was  finally 
purified  by  repeated  recrystallization  from  water.  The 
mother-liquor,  after  the  removal  of  the  saprine  platino- 
chloride, contains  the  mydaleine  salt,  which,  on  account  of 
its  solubility  in  water,  crystallizes  only  on  concentration, 
or  on  standing  under  a  desiccator.  The  mercuric  chloride 
filtrate  contains  some  mydaleine  and  the  ptomaine,  which 
yields  a  platinochloride  containing  28.40  per  cent,  platinum. 

The  free  base  is  a  diamine,  and  was  first  ascribed  the 
formula  C5H16N2.  It  appears,  however,  to  be  isomeric 
with  cadaverine  and  neuridine.  The  term  saprine  is  derived 
from  the  Greek  cairpdg^  signifying  putrid.  It  possesses  a 
weak  pyridine-like  odor,  and  can  be  distilled  with  steam  or 
with  potassium  hydrate  without  undergoing  decomposition. 
In  its  reactions  it  behaves  the  same  as  cadaverine,  except 
that  it  gives  an  amorphous  precipitate  with  potassium- 
bismuth  iodide,  whereas  cadaverine  gives  a  crystalline  pre- 
cipitate. The  free  base  gives  an  immediate  intense  blue 
color  with  ferric  chloride  and  potassium  ferricyanide. 

The  Hydrochloride,  C5H]4N2.2HC1,  forms  flat  needles 
which  are  not  hygroscopic  (distinction  from  cadaverine 
hydrochloride).  Its  reactions  are  the  same  as  those  of 
cadaverine  hydrochloride  (see  Table  I.).  It  is,  however, 
tinged  slightly  blue  by  a  mixture  of  ferric  chloride  and 
potassium  ferricyanide,  whereas  the  free  base  gives  an 
intense  blue.  It  differs  from  cadaverine  in  that  it  does  not 
give  the  reddish-brown  color  with  potassium  bichromate 
and  sulphuric  acid.  Again,  it  forms  no  aurochloride ; 
while,  on  the  other  hand,  cadaverine  hydrochloride  yields 
an  easily  soluble  salt,  crystallizing  in  splendid  needles. 

The  Platinochloride,  C5H14N2.2HCl.PtCl4,  forms 
parallel,  aggregated,  pointed  crystals,  which  are  somewhat 
soluble  in  water,  and  are  thus  distinguished  from  cadaverine 


222  BACTERIAL    POISONS. 

platinochloride,  which  crystallizes  in  rhombs,  and  is  diffi- 
cultly soluble  in  water. 

Physiologically,  it  is  indifferent. 

A  Base,  C7H10W2. — Until  very  recently  the  nature  of  the 
basic  substances  which  are  formed  as  products  of  the  alco- 
holic fermentation  of  sugar  or  molasses  has  been  but  little 
understood.  Kramer  and  Pinner,  in  1869,  found  in  crude 
fusel  oil  a  small  quantity  of  a  volatile  base  which  they 
apparently  identified  with  a  collidine.  This  observation  was 
confirmed  by  Ordonneau  and  others ;  and  still  more  re- 
cently (January,  1888)  Morin  has  contributed  an  elaborate 
paper  upon  the  bases  formed  during  alcoholic  fermentation. 
The  portion  of  crude  fusel  oil  which  boils  above  130.5° 
was  extracted  with  slightly  acidulated  water,  the  acid 
aqueous  solution  thus  obtained  was  made  alkaline,  and  the 
oily  bases  which  were  thus  set  free  were  then  distilled  with 
vapor  of  water.  The  free  bases  were  dried  over  potassium 
hydrate  and  then  subjected  to  fractional  distillation.  Three 
fractions  were  thus  obtained,  boiling  respectively  at  155°— 
160°,  171°-172°,  and  185°-190°,  Only  the  second  frac- 
tion, which  boils  at  171°-172°,  was  studied,  and  was  found 
to  possess  the  formula  C7H10N2.  Heated  with  concentrated 
hydrochloric  acid,  it  is  decomposed  in  part  with  the  forma- 
tion of  ammonia.  It  combines  with  ethyl  iodide  to  form  a 
yellow  crystalline  compound,  which  is  soluble  in  water 
and  alcohol,  insoluble  in  ether.  The  hydrochloride  crys- 
tallizes in  fine  white  needles,  soluble  in  water  and  alcohol, 
and  but  very  slightly  soluble  in  absolute  ether.  The  free 
base,  as  stated  above,  boils  at  171°-172°,  is  very  soluble  in 
water,  alcohol,  ether,  etc.  When  pure  it  forms  a  colorless, 
strongly  refracting,  very  mobile  oil,  which  possesses  a  char- 
acteristic nauseating  odor,  but  slightly  resembling  that  of 
the  pyridine  bases.  Its  density  at  12°  is  0.9826  ;  toward 
litmus  paper  the  base  shows  no  decided  reaction.  The 
platinochloride  is  crystalline  and  is  very  soluble  in  water 
and  alcohol,  slightly  soluble  in  ether.  Potassio-mercuric 
iodide  does  not  precipitate  the  aqueous  solution  of  the  free 
base,  but  in  solutions  of  the  hydrochloride  it  gives  a  yellow 


CHEMISTRY    OF    THE    PTOMAINES.  223 

flocculent  precipitate,  which  soon  crystallizes  in  long  bril- 
liant yellow  needles.  This  reaction  takes  place  readily  in 
solutions  of  1  to  1000,  and  only  after  some  hours  in  solu- 
tions of  1  to  10,000 ;  and  is  not  given  by  the  bases  of  the 
pyridic  and  quinolinic  series.  Mercuric  chloride  produces 
an  immediate  flocculent  precipitate  in  solutions  of  the  base 
having  a  concentration  of  1  to  1000,  but  requires  some 
time  to  appear  in  1  to  10,000.  Phosphotungstic  acid  gives 
an  immediate  white  precipitate  even  in  a  dilution  of  1  to 
10,000.  Phosphomolybdic  acid  in  solutions  of  the  same 
strength  yields  a  yellow  precipitate. 

The  physiological  action  of  this  base  has  been  examined 
by  R.  Wurtz,  who  found  the  lethal  dose  for  rabbits,  etc., 
to  be  about  one  gramme  per  kilogramme  of  body  weight. 
It  produces  stupor,  paralysis,  which  at  first  appears  in  the 
rear  extremities ;  the  sensibility  becomes  diminished  and 
the  pupils  are  dilated  and  unresponsive  to  light ;  the  rate 
of  heart-beat  is  lowered,  and  the  rectal  temperature  falls  as 
low  as  35°  ;  death  follows  a  more  or  less  prolonged  coma. 

Tanret  obtained  by  the  action  of  ammonia  on  glucose 
a  number  of  bases,  to  which  he  applied  the  generic  name 
of  glucosines.  One  of  these,  having  the  formula  C14H10N2 
(C  =  6),  corresponds  in  its  formula  and  its  general  proper- 
ties to  Morin's  base  C7H10Nj  (C  =  12),  and,  in  fact,  the 
two  bases  are  considered  by  Tanret  to  be  identical. 

It  is  interesting  to  note  in  this  connection  that  alkaloidal 
bases  have  been  found  in  petroleum  by  Bandrowski,  and 
that  similar  basic  substances  have  been  detected  by  Weller 
in  paraffin  oil. 

Most  of  the  solvents  in  common  use,  such  as  alcohol, 
ether,  chloroform,  benzole,  petroleum  ether,  amyl  alcohol, 
etc.,  have  been  shown  at  different  times  to  contain  basic 
pyridine  compounds,  though  ordinarily  in  very  minute 
quantity.  On  the  other  hand,  Haitinger  has  found  in 
some  specimens  of  amyl  alcohol  as  much  as  0.5  per  cent, 
of  pyridine. 

Susotoxine,  C10H26N2  ("?),  is  a  base  isolated  by  Now  in 
1890  from  cultures  of  the  ho^-cholera  bacillus  of  Salmon 


224  BACTERIAL    POISONS. 

(swine-plague  of  Billings).  It  is  probably  identical  with 
the  base  obtained  by  v.  Schweinitz  from  the  same  germ, 
although  the  formula  ascribed  to  it  by  him  is  C14H32]Sr2. 
The  free  base  has  not  been  obtained.  The  hydrochloride 
forms  a  light-yellow  syrup  which  shows  no  tendency  to 
crystallize.  It  is  soluble  in  water  and  in  absolute  alcohol, 
and  is  somswhat  hygroscopic.  When  heated  with  fixed 
alkali  it  gives  off  a  strong  amine  odor,  such  as  is  perceived 
on  evaporating  the  original  culture-fluid,  if  it  happens  to  be 
alkaline  in  reaction. 

The  platinochloride  is  obtained  by  precipitation  as  a  light, 
flesh-colored,  granular  precipitate.  It  is  readibly  soluble 
in  water,  from  which  it  can  be  reprecipitated  by  addition 
of  absolute  alcohol.  From  aqueous  solution,  when  allowed 
to  evaporate  slowly,  it  crystalliz3s  in  long,  thick  needles. 

The  mercurochloride  is  thrown  down  from  solutions  of 
the  hydrochloride  in  absolute  alcohol,  by  alcoholic  mercuric 
chloride,  as  a  heavy,  white,  granular  precipitate.  This 
readily  dissolves  on  the  addition  of  a  small  quantity  of 
water,  and  can  be  perfectly  reprecipitated  by  addition  of 
absolute  alcohol.  On  treatment  with  hydrogen  sulphide  it 
is  readily  decomposed,  yielding  the  pure  hydrochloride 

The  aurochloride  is  very  soluble  in  water  and  alcohol. 
From  the  alcoholic  solution  it  may  be  partially  precipitated 
by  ether  as  a  light-yellow,  oily  precipitate,  which  is  adhe- 
rent to  the  sides  and  bottom  of  the  tube. 

Physiological  Action. — The  base  is  toxic  only  in  rela- 
tively large  doses,  as  seen  from  the  following  experiment. 
About  100  milligrammes,  dissolved  in  a  little  water,  were 
injected  subcutaneously  into  a  young  rat.  The  animal 
was  at  first  quiet,  apparently  unwilling  to  move.  After 
some  ineffectual  attempts  at  jumping,  it  settled  down  in  a 
recumbent  position,  and  when  placed  on  its  side  was  unable 
to  rise.  Respiration  was  at  first  retarded,  later  increased, 
but  toward  the  end  was  again  very  slow.  Convulsive  tre- 
mors shook  the  body  at  frequent  intervals.  The  animal 
kicked  vigorously.  Reflexes  were  present  almost  to  the 
end.  As  death  approached,  the  red  eyes  whitened  and  took 
on  a  glazed,  opaque  appearance.     Death  resulted  in   one 


CHEMISTRY    OF    THE    PTOMAINES.  225 

and  a  half  hours.  The  animal  was  on  its  side,  the  feet 
extended.  Post-mortem  examination  showed  the  heart 
arrested  in  diastole,  lungs  rather  pale,  stomach  contracted, 
serum  in  thoracic  cavity,  subcuta  pale  and  cedematous. 
Repeated  doses  of  smaller  quantities  seem  to  confer  a  partial 
immunity  to  the  action  of  the  germ. 

Methyl-guanidine,  C2H7N3,  =NH=  C\^  — ch3- 

This  base  has  long  been  known  as  a  product  of  the  oxi- 
dation of  creatine  and  creatinine,  but  had  never  been  met 
with  in  animal  tissues.  Brieger  in  1886  (III.,  33)  ob- 
tained it  from  horseflesh  which  was  allowed  to  decompose 
in  a  closed  vessel  at  a  low  temperature  ( — 9°  to  -\-  5°)  for 
four  months.  Bockeisch  (Ber.  20,  1441)  isolated  it  from 
impure  cultures  on  beef-broth  of  Finkler  and  Prior's 
vibrio  proteus,  containing  ordinary  putrefaction  bacteria, 
for  twenty  to  thirty  days  at  37°-38°.  Vibrio  proteus 
alone  seems  incapable  of  forming  this  base.  The  comma 
bacillus  after  some  time  (six  weeks)  partially  decomposes 
creatinine  with  formation  of  a  small  cpuantity  of  methyl- 
guanidine  (Brieger).  The  bacillus  of  anthrax  likewise  is 
capable  of  transforming  creatine  into  methyl-guanidine. 

It  occurs  in  the  mercuric  chloride  filtrate  (Brieger), 
from  which  it  is  obtained,  after  the  removal  of  the  mercury 
by  hydrogen  sulphide,  by  precipitation  with  phospho- 
molybdic  acid.  The  precipitate  is  decomposed  with  neutral 
lead  acetate,  and  the  filtrate  from  this,  after  removal  of  the 
lead  by  hydrogen  sulphide,  is  concentrated  and  then  sodium 
picrate  added.  The  resinous  picrate  precipitate  is  purified 
by  boiling  with  much  water,  and,  finally,  it  is  recrystallized 
from  boiling  absolute  alcohol.  According  to  Bocklisch, 
it  occurs  in  the  mercuric  chloride  precipitate  (not  in  the 
filtrate),  from  which  it  is  isolated,  after  removal  of  the  mer- 
cury and  concentration  of  the  clear  filtrate,  by  precipitation 
with  sodium  picrate.  The  precipitate  containing  cadaverine, 
methyl-guanidine,  and  creatinine,  is  boiled  with  absolute 
alcohol  (cadaverine  picrate  is  insoluble)  and  the  alcoholic 
solution  is  then  evaporated  to  drive  off  the  alcohol  and 


226  BACTERIAL    POISONS. 

taken  up  with  water.  From  this  aqueous  solution,  after 
removal  of  picric  acid,  rnethyl-guanidine  is  precipitated  by- 
gold  chloride,  whereas  creatinine  remains  in  solution. 

This  ptomaine  is  identical  with  the  synthetic  methyl- 
guanicline  (methyluramine)  which  can  be  readily  obtained 
by  boiling  a  creatine  solution  with  mercuric  oxide  or  with 
lead  dioxide  and  dilute  sulphuric  acid  (Dessaignes).  The 
parent  substance  of  methyl-guanidine  as  it  occurs  in  putre- 
faction is  undoubtedly  the  creatine  which  exists  preformed 
in  the  muscular  tissue.  If  such  is  the  case,  the  bacteria 
engaged  in  its  production  must  be  considered  as  possessing 
an  oxidizing  action,  since  this  base  is  prepared  synthetically 
from  creatine  by  oxidation.  That  creatine  does  not  oiFer 
much  resistance  to  the  action  of  bacteria  is  shown  in  the 
fact  that  Friedlander's  pneumonia  coccus,  which  pos- 
sesses but  small  chemical  powers,  is  capable  of  slowly  but 
steadily  decomposing  creatine,  yielding  as  one  of  the  pro- 
ducts acetic  acid.  Strecker  aud  Erlenmeyer,  as  well 
as  Baumanjst,  have  shown  that  creatine,  although  a  sub- 
stituted guanidine,  is  not  poisonous,  but  is  readily  converted 
into  creatinine,  which  is  a  relatively  toxic  substance.  On 
the  other  hand,  guanidine  and  methyl-guanidine  are  quite 
violent  poisons.  This  is,  therefore,  another  instance  in 
which  a  toxic  substance  is  formed  by  the  action  of  bacteria 
from  a  previously  non-poisonous  base  (see  page  244). 
According  to  Lossen,  guanidine  is  formed,  though  in 
small  quantity,  in  the  oxidation  of  albumin. 

The  formulas  of  these  closely  related  substances  are  here 
given  for  comparison : 

Creatine,    NH=C<™.CH,C02H 

/N(CH3).CH, 
Creatinine,  NH^Cx^jtt  _      [^ 

/N(CH3).CH2 
Methyl-hydantoine,  0  =  C\^tt pn 

Methyl-guanidine,  NH  =  C<^g  CH* 


CHEMISTKY    OF    THE    PTOMAINES.  227 


Guanidine,  NH  =  Cx  ^tj! 


Urea,0=C<™: 


Methyl-guanidine  forms  a  colorless,  easily  deliquescent 
mass  possessing  a  strong  alkaline  reaction.  On  heating 
with  potassium  hydrate  it  decomposes,  and  yields  ammonia 
and  methylamine.     It  is  a  highly  poisonous  base. 

The  Hydeochloeide,  C2H7N3.HC1,  cau  be  obtained 
from  the  picrate  by  dissolving  the  latter  in  water  acidulated 
with  hydrochloric  acid,  and  extracting  the  solution  with 
ether  to  remove  the  picric  acid.  The  colorless  aqueous 
solution  now,  on  evaporation,  yields  a  thin  syrup  which 
crystallizes  in  vacuum  to  compact  prisms.  These  are  in- 
soluble in  alcohol,  and  give  with  platinum  chloride  a  double 
salt  of  monoclinic  needles  (Haushofee)  which  are  very 
easily  soluble  (1  part  in  about  7  parts  water,  Tataeinow). 

The  Aueochloeide,  C2H7N"3.HCl.AuCl3  (Au  =  47.71 
per  cent.)  forms  rhombic  crystals  (Haushofee)  which  are 
easily  soluble  in  ether,  more  difficultly  in  water  or  alcohol ; 
readily  soluble  (Beiegee).  It  readily  decomposes  on  heat- 
ing in  pure  water,  but  may  be  recrystallized  from  water 
acidulated  with  hydrochloric  acid.     It  melts  at  198°. 

The  Piceate,  C2H7N3.C6H2(N02)3OH,  comes  down  at 
first  as  a  resinous  precipitate,  which  when  boiled  with  much 
water  solidifies  in  the  form  of  felted  needles,  It  is  very 
difficultly  soluble  in  water,  and  can  be  purified  by  repeated 
recrystallization  from  boiling  absolute  alcohol — distinction 
from  cadaverine.     It  melts  at  192°. 

The  Oxalate,  (C2H7N3)2.H2C204+2H2O,  forms  crystals 
which  are  easily  soluble  in  water. 

Physiological  Action. — Methyl-guanidine  as  obtained 
from  putrefying  flesh  is  identical  in  its  physiological 
action  with  the  synthetic  base.  It  has  already  been  stated 
that  the  non-poisonous  creatine  is  readily  converted  into  the 
relatively  energetic  poison  creatinine.  The  latter  substance 
possesses  a  paralyzing  action  differing  very  much  from  its 


228  BACTERIAL    POISONS. 

decomposition-product  methyl-guanidine.  This  base  is  very- 
poisonous,  and  the  symptoms  are  marked  by  dyspnoea, 
muscle  tremor,  and  general  clonic  convulsions.  Brieger 
has  observed  the  following  symptoms  on  injection  of  about 
0.2  gramme  of  methyl-guanidine  into  a  guinea-pig :  The 
respiration  at  once  becomes  more  rapid,  and  in  a  few  min- 
utes abundant  passage  of  urine  and  stool  takes  place ;  the 
pupils  dilate  rapidly  to  the  maximum  and  cease  to  react. 
The  animal  is  uneasy  but  motionless,  though  not  exactly 
paralyzed.  Respiration  becomes  deeper  and  more  labored, 
the  head  moves  from  side  to  side,  the  extremities  become 
gradually  paralyzed  ;  dyspnoea  sets  in,  the  animal  falls  on 
its  side  and  dies  (twenty  minutes)  amid  general  clonic  con- 
vulsions of  short  duration.  Fibrillary  twitchings  of  the 
trunk  muscles  are  observed  only  in  the  beginning.  Post- 
mortem showed  the  heart  to  be  stopped  in  diastole,  the  in- 
testines filled  with  fluid,  the  bladder  contracted,  the  cortex 
of  the  kidney  hyperremic,  but  the  papillae  of  the  kidneys 
surprisingly  pale. 

Morrhuine,  C19H27N3,  was  obtained  by  Gautier  and 
Mourgues  (1888)  from  the  mother  liquors  of  aselline  on 
concentration  of  the  platinum-containing  liquid.  This  sub- 
stance constitutes  about  one-third  (0.07  per  cent.)  of  all  the 
bases  found  in  cod-liver  oil,  and  is  named  from  Gadus 
morrhua,  the  ordinary  codfish.  The  free  base  is  an  oily, 
very  thick,  amber-yellow  liquid,  the  odor  of  which  resem- 
bles somewhat  that  of  syringa.  It  floats  on  water  and  par- 
tially dissolves ;  is  more  soluble  in  ether  and  in  alcohol. 
The  base  is  very  alkaline  and  is  caustic  to  the  tongue.  It 
absorbs  carbonic  acid  and  is  non-volatile.  The  salts  of 
copper  are  precipitated  by  it,  but  the  hydrate  formed  is  not 
redissolved 

The  hydrochloride  is  very  deliquescent.  The  gold  salt 
forms  a  yellow  precipitate  which  readily  dissolves  on 
warming.  The  platinum  salt,  C19H27N3.2HCl.PtCl4  (Pt  = 
27.56  per  cent.),  crystallizes  in  barbed  needles,  which  are 
quite  soluble.     (Separation  from  aselline,  p.  230). 


CHEMISTKY    OF    THE    PTOMAINES.  229 

Physiological  Action. — The  base  possesses  the  property 
of  exciting  the  appetite ;  it  acts  as  a  diaphoretic  and  above 
all  as  a  diuretic.  0.029  gramme  given  subcutaneously 
to  a  guinea-pig  produced  in  two  and  a  half  hours  a  loss 
of  13.5  grammes  in  the  weight  of  the  animal.  The  same 
effect  is  produced  in  birds.  Strong  doses  (0.1  gramme  per 
kilogramme)  produce  fatigue  and  hebetude. 

A  Base,  C13H20N"4,  was  obtained  as  early  as  1868  by 
Oser,  who  observed  its  formation  during  the  fermentation 
of  pure  cane-sugar  by  means  of  yeast.  The  hydrochloride 
when  dried  in  vacuo  is  said  to  form  a  white,  very  hygro- 
scopic foliaceous  mass,  which  soon  becomes  brown  on  expo- 
sure to  air.  At  first  it  imparts  a  burning  taste,  which  is 
soon  replaced  by  a  very  bitter  sensation. 

A  Base  corresponding  to  the  formula  Cl7H38N4  was  ob- 
tained by  Gautier  and  Etard  from  the  mother-liquors  of 
the  platinochloride  of  the  base  C8H13]Sr.  Very  little  is 
known,  however,  in  regard  to  the  general  properties  of  this 
base,  owing  to  the  small  quantity  which  could  be  isolated. 
This  base  and  the  one  obtained  by  Oser  from  the  yeast- 
fermentation  of  sugar,  C13H20N4,  and  aselline,  C25H32N4, 
are  the  only  ptomaines  thus  far  isolated  which  are  known 
to  contain  four  atoms  of  nitrogen. 

The  Platinochloride,  C17H38N4.2HCl.PtCl4  (Pt  = 
27.52  per  cent.),  is  readily  soluble,  and  crystallizes  in 
needles  which  possess  a  light-yellow  flesh  color.  When 
heated  to  100°,  it  slowly  decomposes,  giving  off  a  syringa- 
like  odor. 

Aselline,  C25H32N4,  isolated  by  Gautier  and  Mour- 
gues  (1888),  together  with  five  other  bases  from  cod-liver 
oil.  (See  p.  263.)  It  is  present  only  in  small  quantity  in 
the  oil.  The  name  is  derived  from  Asellus  major,  the  great 
codfish.  The  free  base  is  thrown  dowii  from  the  solutions 
of  the  hydrochloride  by  the  addition  of  alkali,  in  amorphous 
white  floceules  which  are  almost  insoluble  in  water.  It  is 
almost  colorless,  but  on  exposure  to  the  air  becomes  slightly 

11 


230  BACTERIAL    POISONS. 

green.  It  is  not  hygroscopic,  and  possesses  a  density  of  about 
1.05.  On  heating  it  melts  to  a  viscid  yellowish  fluid,  pos- 
sessing an  aromatic  odor  ;  is  non-volatile.  Although  almost 
insoluble  in  water,  it  imparts  to  it  an  alkaline  reaction  and 
a  bitter  taste.     It  is  soluble  in  ether,  more  so  in  alcohol. 

The  salts  are  cry  stall  izable,  but  are  partially  dissociated 
by  the  action  of  warm  water.  The  hydrochloride  forms 
crossed  or  entangled  needles  which  are  quite  bitter.  The 
gold  salt  is  very  reducible.  The  platinochloride,  C25H32N4. 
2HCl.PtCl4  (Pt=  24.41),  is  orange-yellow  in  color;  solu- 
ble in  warm  waler,  insoluble  in  cold  water  (separation 
from  morrhuine,  p.  228),  and  is  rapidly  changed  by  boiling- 
water.  The  mercury  salt  is  precipitated  in  the  cold  ;  redis- 
solves  on  heating,  and  then,  on  cooling,  recrystallizes. 

In  large  doses  it  produces  fatigue,  short  and  rapid  respi- 
ration, and  stupor.  Three  milligrammes  of  the  hydro- 
chloride kills  a  greenfinch  in  fourteen  minutes. 

Mydine,  C8HnNO,  is  a  non-poisonous  base  which 
was  obtained  by  Brieger  in  1886  (III.,  25)  from  the 
putrefaction  of  about  two  hundred  pounds  of  human  in- 
ternal organs  ;  and  also  in  cultures  of  the  Eberth  bacillus 
on  peptonized  blood-serum.  It  occurs  iu  the  mercuric 
chloride  filtrate,  and  is  isolated  from  it  after  the  removal 
of  the  mercury  by  hydrogen  sulphide,  by  precipitation  with 
phosphomolybdic  acid.  The  gummy  precipitate  which  is 
produced  is  decomposed  on  the  water-bath  with  a  solution 
of  neutral  lead  acetate,  and  the  filtrate  on  evaporation  yields 
a  colorless  hydrochloride,  crystallizing  in  plates.  It  is 
purified  by  recrystallization  of  the  picrate. 

The  free  base  is  strongly  alkaline,  and  possesses  an  am- 
moniacal  odor.  It  is  characterized  by  its  strong  reducing 
properties.  The  name  mydine  is  derived  from  fivdau,  to 
putrefy.  With  platinum  chloride  it  gives,  after  a  time,  an 
extremely  soluble  salt ;  with  gold  chloride,  a  precipitate  of 
metallic  gold.     On  distillation  it  is  decomposed. 

The  Hydrochloride,  C8HuNO.HC1,  crystallizes  in 
colorless  plates.  It  gives  a  blue  color  with  ferric  chloride 
and  potassium  ferricyaniele. 


CHEMISTRY    OF    THE     PTOMAINES.  231 

The  Picrate,  C8H1iN0.C6H2(NO2)3OH,  is  obtained  in 
broad  prisms,  which  melt  at  195°.  It  is  the  only  salt 
suitable  for  manipulations. 

In  describing  Nencki's  collidine  (page  196)  it  was  stated 
that  tyrosin  might  be  looked  upon  as  the  source  of  that- 
base.  It  would  seem,  however,  to  be  more  appropriately 
the  parent  substance  of  mydine,  inasmuch  as  it  decomposes 
on  being  heated  to  270°  into  carbonic  acid  and  oxyphenyl- 
ethylamine,  CgH^NO.  The  change  that  takes  place  can 
be  represented  by  the  equation  : 

C«H<<Ch2.CHNH2.C02H  =  0A<g£.0H^H,  +  cor 

Tyrosin.  Oxypiienyl-ethylamine. 

A  Base,  C5HuN02,  was  isolated  by  E.  and  H.  Sal- 
kowski  (1883)  from  decomposing  fibrin  and  meat.  In  its 
composition  it  is  isomeric  with  betaine  anhydride.  It  is 
extremely  soluble  in  water,  very  difficultly  so  in  alcohol, 
insoluble  in  ether,  and  possesses  a  semen-like  odor  and 
saline  taste.  The  aqueous  solution,  which  is  not  alkaline 
in  reaction,  yields  on  evaporation  a  stellate  crystalline  mass, 
which  on  standing  over  sulphuric  acid  becomes  a  white 
powder,  which  melts  at  156°.  It  dissolves  silver  oxide, 
but  not  cupric  hydrate,  thus  apparently  indicating  that  it- 
is  not  an  amido  acid.  Moreover,  it  does  not  give  a  pre- 
cipitate or  blue  coloration  with  copper  acetate,  or  ammo- 
niacal  silver  nitrate.  It  thus  differed  from  the  then  known 
amido-valerianic  acids,  its  isomers.  Recently,  however 
(1891),  Gabriel  and  Aschan  showed  that  ^-amido-vale- 
rianic acid  agrees  with  this  base  in  its  reactions  to  copper 
and  silver  oxide,  copper  acetate,  and  ammoniacal  silver 
nitrate.  The  gold  salt  of  the  synthetic  base  possessed  the 
same  composition  as  that  of  Salkowski,  and  melted  at 
86°-87°. 

The  identity  of  this  base  with  ^-amido-valerianic  acid 
(homopiperidinic  acid)  would  seem  to  be  established,  and 
as  such  it  is  regarded.  Its  structure,  then,  is  represented  by 

NII2.CH2.CH2.CH2.CH2.C02H. 


232  BACTERIAL    POISONS. 

For  its  synthetic  preparation  see  Ber.  24,  1365  (1891). 
The  base  does  not  seem  to  possess  a  toxic  action. 

The  Hydrochloride,  C5HuN02.HC1,  forms  colorless, 
stellate  crystals,  which  are  permanent  in  the  air,  and  are 
extremely  soluble  in  water,  even  in  absolute  alcohol. 

The  Aurochloride,  C5HuN02.HCl.AiiCl3  +  H20,  is 
obtained  on  slow  evaporation,  as  large,  well-formed,  beau- 
tiful dark-yellow  crystals.  They  are  probably  monoclinic, 
contain  water  of  crystallization,  and  melt  at  below  100°. 

The  Platinochloride  gave  on  analysis  results  cor- 
responding to  the  formula  (C7H15N02.HCl)2PtCl4.  This 
may  possibly  be  due  to  the  presence  of  some  higher  homo- 
logues  of  the  base  C5HuN02.  It  forms  fine  orange-yellow 
crystals,  which  are  very  difficultly  soluble  in  alcohol,  easily 
so  in  hot  water,  from  which,  on  cooling,  it  crystallizes  in 
beautiful  plates. 

Choline  Group. — The  following  four  bases  are  closely 
related,  and,  indeed,  starting  from  choline,  the  oldest  and 
best-known  individual,  the  remaining  bases  can  be  readily 
prepared  from  it.  Moreover,  they  can  all  be  prepared 
synthetically  according  to  methods  that  will  be  subsequently 
indicated.  As  choline  is  the  most  prominent  member,  we 
have  thought  best  to  class  these  substances  together  as  con- 
stituting the  choline  group.  It  is  very  probable  that  my- 
datoxine  and  mytilotoxine,  when  their  constitution  becomes 
known,  will  be  found  to  be  homologues  of  certain  members 
of  this  group. 

Neurine,  C5H13NO  =  C2H3.N(CH3)3.0H.  — This  sub- 
stance was  obtained  and  named  thus  by  Liebreich  (1865), 
who  prepared  it  by  boiling  protagon  for  twenty-four  hours 
with  concentrated  baryta,  Previous  to  its  discovery  as  a 
decomposition-product  of  protagon  from  the  brain  it  was 
prepared  synthetically  by  Hofmann  (1858)  by  treating 
trimethylamine  and  ethylene  bromide  with  potassium  hy- 
drate or  silver  oxide.  Baeyer  (1866),  by  boiling  an  alco- 
holic extract  of  the  brain  with  baryta  water,  obtained  on 
separation  by  three  different  methods,  a  base,  or  rather  a 


CHEMISTKY    OF    THE    PTOMAINES.  233 

mixture  of  bases,  which,  on  analysis,  gave  results  corre- 
sponding to  the  three  formula? : 

1  2  3 

(C5HuNOCl)./PtCl4        (05HuN01)aPtCI4        (0BHuNCl)aP't014 

Formula  No.  3  was  the  one  accepted  by  Ltebeeich  for 
neurine,  but,  according  to  Baeyer,  Liebreich's  neurine 
salt  is  not  simple,  but  is  a  mixture  of  Nos.  1  and  2.  He 
himself  accepts  formula  No.  1  as  the  platinochloride  of 
neurine,  and  distinctly  states  (Annul,  d.  Chem.  u.  Pharm., 
142,  323,  18(37)  that  neurine  is  in  composition  trimethyl- 
oxyethyl-ammonium  hydroxide.  And,  according  to  him, 
choline  from  bile,  and  sinkaline  from  white  mustard,  appear 
to  be  identical  with  neurine. 

This  nomenclature  of  Baeyer's  was  at  first  adopted  by 
Wurtz  and  others,  who  showed  that  the  oxyethyl  base 
was  identical  with  choline  and  sinkaline.  On  that  account 
Strecker,  in  1868  (AnnaL,  148,  79),  suggested  the  re- 
striction of  the  name  choline  to  the  oxyethyl  base,  and  to 
reserve  the  name  neurine  for  the  base  whose  platinochloride 
is  represented  in  No.  3,  as  originally  was  done  by  Lieb- 
reich.  In  1869  Liebreich  showed  conclusively  that 
pure  protagon,  when  heated  witli  baryta  for  twenty-four 
hours,  yields  a  substance  having  the  composition  of  the 
vinyl  base : 

N(CH3)3.C2H3.OH. 

The  platinoahloride  of  this  base  crystallized  in  five-sided 
yellow  plates,  which,  after  a  time,  on  exposure  to  the  air, 
became  cloudy ;  on  treatment  now  with  water  a  portion 
dissolved,  and  the  solution  was  found  to  contain  the  oxy- 
ethyl base.  Furthermore,  he  observed  that  when  the  alco- 
holic extract  of  the  brain,  from  which  all  the  protagon  had 
been  removed,  is  treated  with  baryta,  only  the  latter,  the 
oxyethyl  base,  is  obtained.  Finally,  in  1870,  Wurtz 
abandoned  the  use  of  the  term  neurine  to  designate  the 
oxyethyl  base,  and  returned  to  the  name  choline,  originally 
applied  to  the  oxyethyl  base  by  its  discoverer,  Strecker. 
Nevertheless,  the  confusion  in  the  use  of  these  two  terms 


234  BACTEKIAL    POISONS. 

continued  to  exist,  and  even  at  the  present  time  it  is  the 
cause  of  no  little  misunderstanding.  Thus,  Marino-Zuco 
(1885),  in  his  excellent  researches  on  the  genesis  of  pto- 
maines, applies  the  term  neurine,  following  Baeyer's  pre- 
cedent, to  the  oxyethyl  base,  C5H15N02,  which  is  really 
choline,  according  to  the  proper  nomenclature. 

We  have  gone  somewhat  at  this  point  in  detail  into  the 
history  and  the  proper  use  of  the  terms  neurine  and  choline 
because  of  the  coufusion  which  is  sure  to  arise  if  the  dis- 
tinction is  not  thoroughly  borne  in  mind.  The  name 
neurine,  then,  should  be  used  only  to  denote  the  vinyl  base 
C5H13NO.  It  is  trimethyl-vinyl-ammonium  hydrate.  On 
the  other  hand,  choline  is  applied  to  the  oxyethyl  base 
C5H15N02,  which  is  trimethyl-oxy ethyl-ammonium  hydrate. 

Neurine  has  been  obtained  by  Brieger  (1883)  in  the 
putrefaction  of  horse,  beef,  and  human. flesh  for  five  to  six 
days  in  summer.  It  also  occurs  in  the  commercial,  so-called 
"  neurine,"  together  with  choline  (Brieger,  I.,  31).  Lieb- 
reich  obtained  it  in  the  decomposition  of  protagon  by 
baryta.  And  Brieger  (I.,  60)  also  has  isolated  it  along 
with  choline  from  fresh  human  brains,  by  boiling  with 
baryta ;  but  has  not  obtained  it  by  digesting  the  brains  on 
the  water-bath  with  two  per  cent,  hydrochloric  acid.  It 
has  been  found  in  putrid,  and  as  result  of  this  change 
poisonous,  mushrooms  (Berlinerblau,  1888). 

The  genesis  of  neurine  is  still  rather  obscure,  and  it  is 
to  be  hoped  that  future  investigations  may  shed  more  light 
upon  the  mysterious  production  of  this  highly  poisonous 
base.  Its  occurrence  in  the  brain  together  with  choline 
would  seem  to  indicate  that  it  is  either  derived  from 
choline  by  the  removal  of  water,  or  that  it  exists  together 
with  choline,  partly  replacing  the  latter  in  the  molecule  of 
protagon  (lecithin),  according  to  the  hypothesis  put  for- 
ward by  Lippmann  (page  241).  The  question  of  its  ^ 
derivation  from  choline  by  withdrawal  of  a  molecule  of 
water  has  already  been  subjected  to  an  interesting  experi- 
mental discussion.  Ch.  Gram  attempted  to  explain  the 
production  of  neurine  and  other  musearine-like  ptomaines 
as  due  to  the  dehydrating  action  of  the  acids  employed  in 


CHEMISTRY    OF    THE    PTOMAINES.  235 

the  methods  of  extraction,  and,  indeed,  he  claimed  to  have 
converted  choline  platinochloride,  by  heating  with  hydro- 
chloric acid,  into  nenrine.  This  statement  has  been  dis- 
puted by  Brieoer,  who  showed  that  the  platinochloride 
of  choline,  as  well  as  the  hydrochloride,  may  be  heated 
with  fifteen  or  thirty  per  cent.,  or  even  concentrated  hydro- 
chloric acid,  for  six  to  eight  hours  on  a  water-bath,  with- 
out any  conversion  whatever  (III.,  15).  That  neurine 
may  be  obtained  from  choline,  at  least  by  chemical  pro- 
cesses, was  shown  by  Baeyer,  in  1866,  who  found  that 
choline  chloride,  when  heated  with  several  times  its  volume 
of  concentrated  hydriodic  acid  and  some  red  phosphorus, 
gave  a  compound  C5H13NI2  which,  on  digestion  with  fresh, 
moist  silver  oxide,  yielded  a  vinyl  base  identical  with  that 
previously  obtained  synthetically  by  Hofmann,  and  now 
known  as  neurine.  Brieger  has  tried,  unsuccessfully,  to 
bring  about  this  dehydration  by  the  putrefaction  of  pure 
choline  (I.,  59).  However,  Schmidt  and  Weiss  (1887) 
were  more  successful,  and  they  found  that  choline,  as  well 
as  the  hydrochloride  and  lactate,  is  changed  by  the  action 
of  microorganisms  into  the  strongly  poisonous  neurine. 
Their  results  are  given  in  full  under  choline  (see  page  244.) 
From  what  has  been  said  it  is  evident  that  neurine  can 
only  arise  from  choline,  and  this,  as  will  be  seen  later,  is 
derived  from  lecithin. 

Neurine  is  almost  invariably  accompanied  by  choline, 
from  which,  however,  it  can  be  readily  separated  by  the 
difference  in  the  solubilities  of  the  platinochlorides.  It 
occurs  in  the  mercuric  chloride  precipitate  (and  iu  the 
filtrate);  and  from  this  it  can  be  obtained,  after  removal  of 
the  mercury,  by  precipitating  the  solution  of  the  mixed 
hydrochlorides  in  absolute  alcohol  by  platinum  chloride. 
The  platinochlorides  are  then  separated  by  recrystallization 
from  water,  since  the  neurine  is  difficultly  soluble,  while 
the  choline  salt  is  readily  soluble. 

The  free  base  possesses  a  strong  alkaline  reaction,  and 
on  contact  with  the  fumes  of  hydrochloric  acid  it  yields  a 
cloud.  According  to  Liebreich,  the  alkaline  solution 
cannot  be  neutralized  by  passing  through  it  carbonic  acid. 


236  BACTERIAL    POISONS. 

The  Chloride,  C5H12N.C1,  is  extremely  poisonous,  and 
crystallizes  in  fine  hygroscopic  needles. 

The  Platinochloride,  (CgH12N.Cl)2PtCl4  (Pt  =  33.60 
per  cent.),  is  difficultly  soluble  in  hot  water,  and  crystallizes 
in  beautiful,  well-formed  octahedra  belonging  to  the  regular 
system.  No  twin-crystals  are  observed.  Sometimes  the 
crystals  contain  water  of  crystallization,  at  other  times  they 
do  not  (Brieger,  I.,  33).  According  to  Liebreicii,  it 
forms  from  an  aqueous  solution  in  five-  or  six-sided,  heaped- 
up  plates  resembling  urea  nitrate,  while  from  an  alcoholic 
solution  it  forms  needles,  which  on  exposure  to  air  become 
opaque,  and  are  partially  converted  into  the  oxyethyl  base 
— choline. 

The  Aurochloride,  C5H12N.Cl.AuCl3  (An  =  46.37 
per  cent.),  forms  flat  prisms,  which  are  difficultly  soluble 
in  hot  water  (Brieger.)  Dissolves  easily,  and  can  be 
purified  by  crystallization  (Liebreich). 

Physiological  Action. — Neurine  is  exceedingly  poisonous, 
even  in  small  doses,  and  in  its  action  it  strongly  par- 
takes of  the  characteristic  stamp  of  poisoning  by  muscarine. 
The  injection  of  a  few  milligrammes  into  frogs  produces 
in  a  short  time  a  complete  paralysis  of  the  extremities,  with 
deadening  of  reflex  excitability.  Respiration  stops  first, 
while  the  rate  of  heart-beat  gradually  decreases  till,  finally, 
stoppage  in  diastole  takes  place.  The  injection  of  atropine 
at  this  point  does  away  with  the  effect  of  neurine,  so  that 
the  heart  begins  to  beat  again.  Previously  atropinized 
frogs,  as  a  rule,  withstand  the  action  of  the  poison.  Im- 
mediately after  the  introduction  of  this  substance  there  can 
be  observed  a  distinct  period  of  exaltation,  which,  however, 
soon  gives  way  to  the  characteristic  stage  of  depression  seen 
in  the  progressive  slowing  of  the  rate  of  heart-beat.  Of 
the  warm-blooded  animals,  cats  seem  to  be  much  more 
sensitive  to  its  action  than  mice,  rabbits,  or  guinea-pigs. 
The  symptoms  seen  in  rabbits  are  profuse  moistening  of  the 
nasal  cavities  and  upper  lip,  which  is  succeeded  by  an  in- 
tensely profuse  salivation ;  later  on  there  is  noticeable  an 
abundant  secretion  from  the  nasal  mucous  membrane  and 
from  the  eyes ;  the  latter,  however,  ceases  in  a  short  time. 


CHEMISTRY    OF    THE    PTOMAINES.  237 

The  movements  of  the  heart  and  of  respiration  are  at  first 
quickened  and  strengthened,  but  before  long  the  paralytic 
effects  produce  a  constant  slowing  and  weakening,  till 
finally  complete  cessation  of  both  movements  results.  The 
decided  dyspnoea  observed  gradually  alters  its  character, 
and  just  before  death  the  respiration  is  irregular  and  super- 
ficial. The  heart,  as  in  frogs,  continues  to  beat  after  the 
respiratory  movemeuts  have  ceased,  until  finally  it  stops  in 
diastole.  Direct  application  of  concentrated  solutions  of 
the  poison  to  the  eyes  produces  almost  always  a  contraction 
of  the  pupil,  while  a  similar  but  less  constant  contraction 
is  seeu  when  it  is  injected.  The  peristaltic  action  of  the 
intestines  is  heightened  to  such  an  extent  that  continual 
evacuation  takes  place.  Just  before  death,  violent  clonic 
convulsions  occur.  Atropine  possesses  a  strong  antagonistic 
action  toward  neurine,  and  the  injection  of  even  a  small 
quantity  is  sufficient  to  dispel  the  symptoms  just  de- 
scribed. 

Choline,  C5H15N02  =  C2H4OH.N(CH3)3.OH.  — This 
base  is  identical  with  the  sinkaline  of  VON  Babo,  the  bili- 
neurine  of  Liebreich,  and  the  neurine  of  Baeyer, 
Marino-Zuco,  and  others.  According  to  Schmiedeberg 
and  Harnack,  it  is  identical  with  Letellier's  amanitine 
(agaricine),  to  which  they  assign,  however,  the  formula 
(CH3)3N.(CHOH.CH3)OH.  Choline  was  first  prepared, 
and  so  named,  by  Strecker,  in  1862,  by  treating  hog-bile 
with  hydrochloric  acid.  It  was  prepared  synthetically  by 
Wurtz  (1868)  by  direct  union  of  ethylene  chlorhydrine 
and  trimethylamine.  The  reaction  that  takes  place  can  be 
represented  by  the  equation  : 

CTT    ^  C-H-3  ] 

c*Mc?  +  ™;  u=    gH3.NC1 

u±±3  }  C2H4.OHJ 

Baeyer  (1866)  obtained  it  by  boiling  an  alcoholic  extract 
of  the  brain  with  baryta  water;  and  Liebreich,  in  1869, 
showed  that  if  the  alcoholic  extract,  from  which  all  the 

11* 


238  BACTERIAL    POISONS. 

protagon  had  been  removed,  be  thus  treated,  only  choline  is 
formed,  whereas  pure  protagon,  on  heating  with  baryta, 
yields  neurine.  It  has  been  obtained  from  the  yelk  of 
eggs  ;  from  bile;  from  fresh  brains  (Brieger)  ;  from  fresh 
eggs,  blood,  lungs,  and  hearts,  and  from  lecithin  (Marino- 
Zcjco)  ;  from  human  placenta  (Boehm)  ;  from  the  eye ; 
from  commercial  neurine  (Brieger)  ;  from  fresh  as  well  as 
decomposing  internal  organs  of  the  cadaver  (Brieger, 
1885) ;  from  herring-brine  and  decomposing  pike,  three 
days  in  midsummer  (Bockltsch).  It  has  also  been  isolated 
from  cultures  of  vibrio  proteus  (Bocklisch),  and  of  comma 
bacillus  (Brieger).  Ehrenberg  (1887)  found  it  in 
poisonous  sausage,  and,  by  growing  a  bacillus  obtained  from 
this,  on  liver. 

Not  only  has  choline  been  met  with  in  the  animal  tissues, 
but  it  has  also  been  observed  within  the  last  few  years  to  be 
very  widely  distributed  in  the  vegetable  kingdom,  especi- 
ally so  in  fatty  seeds.  Thus,  it  has  been  found  (Harnack, 
1876)  accompanying  muscarine,  in  toadstool  ( Agaricus  mus- 
carins) ;  in  hops,  and  hence  in  beer  (Griess  and  Harrow  ; 
in  the  seeds  of  Trigonella,  in  Indian  hemp,  areca-  and  earth- 
nuts,  hemp  seeds  and  lentils  (Jahns);  in  the  seeds  of  white 
mustard,  as  a  glycoside  (von  Babo);  in  ergot  (Brieger); 
in  the  germs  of  pumpkins  and  lupines  (Schulze,  Zeitschr. 
f.  Physiol.  Chem.,  11,  365) ;  in  beech-nuts  and  morels  (Hel- 
vellaesculenta,  Boletus  lusiclus,  Amanita  pantherina,  Bohm)  ; 
in  flores  sambuci  (elder),  and  extracts  of  belladonna,  hyos- 
cyamus,  ipecacuanha  root  and  Acorus  calamus  (Kunz), 
and  Scopolia  Japonica  (Schmidt  and  Henschke)  ;  in  the 
sprouts  and  cotyledons  of  Soja  beans  (Schulze,  1888),  in 
the  fat  from  hog's  bean,  vetch,  peas  and  lupines  (Jacob- 
son,  1889)  ;  from  the  lecithin  of  lupine  seeds  (Schulze  and 
Steiger)  ;  and  in  Cheken  leaves  (Myrtus  cheken,  Weifs). 
According  to  Lippmann  {Ber.  20,  3206),  it  is  present,  to- 
gether with  betaine,  in  the  molasses  from  beet-root  sugar. 
Choline  (Ritthausen)  and  betaine  (Bohm)  exist  together 
in  cotton-seeds ;  hence,  choline  occurs  in  the  press-cakes  from 
cotton-seeds  (Bohm).  According  to  Schulze,  and  also 
Ritthausen,  choline   occurs  with   betaine   and   another 


CHEMISTRY    OF    THE    PTOMAINES.  239 

base  in  the  seed  of  the  vetch,  and  in  peas  with  a  base  re- 
sembling betaine.  The  two  bases  have  also  been  found 
together  in  Scopolia  atropo'ides  by  Siebert. 

Choline  may  readily  be  prepared,  after  the  method  of 
Diakonow,  from  the  yelk  of  eggs.  These  are  extracted 
with  ether,  then  with  alcohol,  and  the  extracts  thus  ob- 
tained evaporated,  when  the  resulting  residues  are  boiled 
with  baryta  for  one  hour.  The  filtrate,  after  the  removal 
of  the  barium  by  carbonic  acid,  is  evaporated  and  the 
residue  is  abstracted  with  absolute  alcohol.  The  alcoholic 
solution  is  now  precipitated  with  platinum  chloride. 
Brieoer  (II.,  55)  has  presented  a  method  which  is  much 
simpler  in  its  details  and  obviates  the  use  of  the  expen- 
sive platinum  chloride.  The  tissues  rich  in  lecithin,  as 
yelk  of  egg,  braiu,  etc.,  are  heated  with  concentrated 
hydrochloric  acid  for  some  hours  on  the  water-bath.  The 
insoluble  residue  is  filtered  off,  and  the  filtrate,  after  neu- 
tralization of  the  excess  of  free  acid  with  carbonate  of 
sodium,  is  evaporated.  The  residue  is  extracted  with 
alcohol,  and  the  alcoholic  solution  is  precipitated  with 
alcoholic  mercuric  chloride.  The  precipitate  thus  obtained 
on  recrystallization  several  times  from  a  large  quantity  of 
boiling  water,  yields  the  pure  double  salt  of  choline. 

If  desirable,  it  can  be  made  from  pure  lecithin,  best  pre- 
pared according  to  Gilson's  method.  Yelk  of  eggs  is 
repeatedly  shaken  up  with  ether  until  the  latter  is  colored 
only  a  faint  yellow ;  the  ether  solution  then  distilled,  the 
residue  taken  up  in  petroleum  ether  and  filtered.  The 
filtrate,  in  a  separately  funnel,  is  well  shaken  with  75 
per  cent,  alcohol,  and  this  is  repeated  several  times  with 
fresh  alcohol.  The  alcoholic  extracts  are  combined,  allowed 
to  stand  for  some  time,  then  filtered  and  subjected  to  dis- 
tillation to  remove  traces  of  petroleum  ether.  The  solu- 
tion is  now  set  aside  in  a  cool  place  for  several  days ;  the 
precipitate  which  forms  consists  of  cholesterine,  etc.,  and 
a  little  lecithin.  The  alcoholic  solution  is  filtered  by  de- 
cantation,  then  decolored  by  boiling  with  bone-black ; 
rapidly  evaporated  at  50—60°  to  a  syrupy  consistency. 
This  residue  is  extracted  with  ether,  the  solution  filtered 


240  BACTERIAL    POISONS. 

and  evaporated.  The  lecithin  thus  obtained  is  almost  per- 
fectly pure,  but  contains  traces  of  cholesterine.  To  com- 
pletely purify  it,  it  can  be  dissolved  in  as  little  absolute 
alcohol  as  possible,  and  set  aside  to  reprecipitate  in  the 
cold,  —5  to  15°. 

In  regard  to  the  genesis  of  choline  the  preponderance  of 
testimony  goes  to  show  that  it  is  derived  from  the  decom- 
position of  lecithin,  which,  according  to  the  researches  of 
Diakonow  and  others,  is  one  of  the  most  widely  distributed 
compounds,  occurring  in  greater  or  less  quantity  in  all  of 
the  animal  tissues.  Lecithin,  which  is  a  complex  esther 
(Strecker,  Hundeshagen,  Gilsox),  decomposes  under 
the  action  of  acids  and  alkalies  into  a  base  (choline) 
glycerin,  phosphoric  acid,  and  fatty  acids  (stearic,  oleic, 
palmitic,  etc.).  Gilson  has  shown  that  dilute  sulphuric 
acid  slowly  decomposes  lecithin,  forming  choline,  which, 
after  a  few  days,  disappears ;  on  the  other  hand,  sodium 
hydrate,  in  even  1  per  cent,  solution,  rapidly  decomposes 
it.  This  change  is  undoubtedly  accomplished  in  a  similar 
manner  through  the  agency  of  bacteria.  Brieger  (II., 
17)  is  inclined  to  believe  that  choline  exists  preformed  in 
the  various  tissues,  inasmuch  as  he  has  been  unable  to  ob- 
tain it  from  the  brain,  which  is  rich  in  lecithin,  by  boiling 
with  2  per  cent,  hydrochloric  acid.  (See  Schulze,  page 
242.)  Prolonged  heating  with  concentrated  hydrochloric 
acid  was  necessary  in  order  to  obtain  any  choline  from  the 
brain.  This  result  of  Brieger's  is  somewhat  at  variance 
with  that  of  Marino-Zuco  (see  JRelazione,  etc.,  pages  29, 
30,  and  38),  who  obtained  from  25  grammes  of  lecithin,  by 
the  method  of  Stas,  a  small  quantity  of  the  aurochloride 
of  a  base,  while  from  a  similar  amount  he  obtained  more 
relevant  quantities  by  the  method  of  Dragendorff. 

The  occurrence  of  choline  in  the  vegetable  kingdom 
would  be  inexplicable  to  us  at  present  were  it  not  that 
we  now  know  of  the  existence  of  lecithin-like  bodies  in 
plants,  from  the  decomposition  of  which  substantially  the 
same  products  are  obtained  as  from  the  lecithin  obtained 
from  the  animal  tissues.  The  existence  of  such  a  body  in 
plants  was  first  predicted  by  Scheibler  in  1870,  who  was 


CHEMISTRY    OF    THE    PTOMAINES.  241 

led  to  this  conclusion  in  his  celebrated  study  of  beet-root 
sugar,  because  of  the  presence  of  oleic  acid,  glycerin,  phos- 
phoric acid,  and  betaine,  as  well  as  cholesterin,  in  the  beet- 
root extracts.  This  hypothesis  was  confirmed  by  Hoppe- 
Seyler,  who,  in  1879,  found  a  lecithin  substance  in  yeast. 
Schulze  found  a  similar  .compound  in  the  cotyledons  of 
lupine,  while  Jacobson  observed  its  presence  in  mustard- 
seeds,  in  fenugreek-seeds,  in  maize  and  wheat,  in  the  fat 
from  beans,  peas,  vetch,  and  lupines.  Heckel  showed  its 
presence  in  globularia,  and  Lippmann  has  found  it  in  beet- 
root. According  to  Hoppe-Seyler,  this  lecithin-like  sub- 
stance exists  in  all  vegetable  cells  undergoing  development. 
Schulze  and  Likiernik  (1891)  were  the  first  to  prepare 
lecithin  in  a  pure  condition  from  plants.  It  was  found  to 
possess  the  same  properties  and  yield  the  same  decomposi- 
tion-products as  lecithin  from  animal  tissues.  Up  to  the 
present  time  lecithin  has  always  been  supposed  to  contain  a 
radical,  which  gives  rise  to  choline  on  saponification,  as  an 
essential  component,  while  on  the  other  hand  the  fatty 
acids  entering  its  molecule  are  well  known  to  be  replaceable 
by  one  another.  Thus  we  may  have  a  di-stearine  lecithin 
as  well  as  a  di-oleine  lecithin.  The  existence  of  several 
lecithins  in  the  yelk  of  eggs  has  been  recognized  for  some 
time,  and  according  to  Schulze  and  Likiernik  this  is 
also  true  of  the  lecithins  in  plants.  Recent  observations 
of  Lippmann  (Ber.  20,  3206)  show  that  the  above  basic 
radical,  hitherto  regarded  as  constant  in  lecithin,  may  pos- 
sibly be  capable  of  replacement  by  other  similar  radicals. 
He  found  on  saponifying  with  baryta  two  different  speci- 
mens of  lecithin,  both  obtained  from  beet-root,  that  while 
one  of  them  yielded  oleic  acid,  glycerin,  phosphoric  acid, 
and  betaine;  the  other  lecithin  gave  oleic  acid  (and  some 
other  fatty  acids),  glycerin,  phosphoric  acid,  and  choline, 
with  no  betaine — at  least  not  in  isolable  quantity.  This 
remarkable  difference  has  led  Lippmann  to  suggest  an  ex- 
planation which,  while  it  may  not  be  the  correct  one,  never- 
theless possesses  a  high  degree  of  probability.  According 
to  him,  the  lecithin  molecule  may  contain  interchange- 
able basic  radicals  in  the  same  manner  that  it  contains 


242  BACTERIAL    POISONS. 

interchangeable  acid  radicals.  This  view  is  supported  not 
only  in  the  case  of  beet- root,  where  choline  and  betaine 
exist  together,  but  the  same  two  bases  have  been  observed 
in  cottou-seeds.  A  similar  coexistence  was  observed  in  the 
toad-stool  (Agaricus  muscarius),  in  which  choline  aud  mus- 
carine were  found.  Aud,  lastly,  the  same  condition  holds 
true  probably  for  mytilotoxine  aud  betaine,  which  were 
shown  to  be  present  together  iu  poisouous  mussels. 

Lecithin  cannot  always  be  regarded  as  the  source  of 
choline  in  plants,  since  this  base  is  known  to  occur  as  a 
glucoside  in  the  seeds  of  white  mustard.  The  sinapin  de- 
composes according  to  the  equation  : 

C16H23NOs  +  2H20  =  C5H15N02  +  CnH1205. 

Sinapin.  Choline.  Sinapic  Acid. 

According  to  Schulze  (1891)  the  choline  which  is  iso- 
lated from  pea-  and  vetch-seeds  exists  preformed  in  the 
seeds,  and  does  not  result  from  lecithin  by  the  process  of 
extraction.  This  is  also  probably  true  with  reference  to 
cottonseed-cake.  The  condition  in  which  betaine  exists  is 
not  determined. 

The  protoplasm  itself  is  another  possible  source  of  choline 
as  well  as  of  other  nitrogenous  bases,  as  xanthine,  etc.  We 
know  from  Drechsel's  brilliaut  investigation  (1890)  that 
casein  on  treatment  with  hydrochloric  acid  and  stannous 
chloride  yields  ammonia,  amido  acids,  and  organic  bases — 
lysatine,  C6H13]N"302,  and  lysatinine,  C6HuN30  —  homo- 
logues  of  creatine,  C4H9N302,  and  creatinine,  C^H^N^O. 
From  lysatinine  urea  can  be  readily  obtained  by  treatment 
with  baryta.  Subsequently,  Siegfried  (1891)  showed  that 
vegetable  protoplasm  (conglutin  from  lupine)  when  treated 
iu  the  same  way  yields  similar  products.  Later,  Schulze 
demonstrated  that  the  base,  arginine,  C6H14N402,  is  formed 
iu  lupine  sprouts  at  the  expense  of  the  proteids  present,  and 
he  pointed  out  that  this  base  is  probably  related  to  lysatine, 
from  which  it  differs  only  by  NH  (see  next  chapter). 


CHEMISTRY    OF    THE    PTOMAINES.  243 

Decompositions  of  Choline. — Baeyer  (1866)  suc- 
ceeded in  converting  choline  into  neuriue  by  a  purely 
chemical  process.  This  was  accomplished  by  heating 
choline  chloride  with  concentrated  hydriodic  acid  and 
red  phosphorus  in  a  sealed  tube  at  120°-150°,  whereby 
the  compound  C5H13NI2  was  formed.  The  iod-iodide  of 
choline  thus  obtained,  on  treatment  with  moist  silver  oxide, 
gave  a  base  whose  platiuochloride  corresponded  to  the 
formula  (C5H12N01)2PtCl4  +  H2O.  This  double  salt,  ac- 
cording to  Baeyer,  is  readily  soluble  in  water,  and  gives 
reactions  similar  to  choline.  Although  Baeyer  is  em- 
phatic in  his  assertion  that  this  is  the  vinyl  compound 
(neurine)  formed  from  the  oxy-ethyl  base  (choline),  yet  it 
seems  that  there  is  room  for  doubt  in  regard  to  the 
interpretation  of  his  results.  Thus  neurine  platiuochloride 
is  difficultly  soluble  in  water,  contrary  to  the  behavior  of 
the  platiuochloride  obtained  by  him.  On  the  other  hand, 
choline  platiuochloride  is  easily  soluble  in  water,  and  it 
would  seem,  therefore,  that  Baeyer  has  not  converted 
choline  into  neurine,  but  rather  has  regenerated  choline 
from  its  iod-iodide.  If  such  were  the  case,  we  would  ex- 
pect that  the  iod-iodide  of  neurine,  C5H13NI2,  which  has 
the  same  composition  as  the  corresponding  derivative  of 
choline,  would  yield,  on  treatment  with  silver  oxide,  the 
oxy-ethyl  base.  Baeyer  has  apparently  not  been  able  to 
effect  this  change,  since  he  holds  that  the  vinyl  base  may 
be  prepared  from  the  oxy-ethyl,  but  that  the  reverse,  the 
preparation  of  the  oxy-ethyl  base  from  the  vinyl  compound, 
cannot  be  accomplished. 

Whether  the  change  described  by  Baeyer  takes  place  or 
not,  it  is,  nevertheless,  certain  that  choline  does  not  readily 
give  up  a  molecule  of  water  and  thus  become  converted 
iuto  neurine.  Gil.  Gram  announced,  in  1886,  that  choline 
chloride  and  lactate  on  heating  ou  the  water-bath  de- 
compose, aud  that  this  conversion  into  the  vinyl  base  was 
complete  when  the  aqueous  hydrochloric  acid  solution  of 
choline  platinochloride  was  heated  for  five  or  six  hours  on 
the  water-bath.  In  this  way  Gram  endeavored  to  explain 
the  formation  as  due  to  the  action  of  acids  upon  choline, 


214  BACTERIAL    POISONS. 

but  Brieger  has  shown  that  the  platinum  salt  of  choline, 
as  well  as  its  hydrochloride,  can  be  heated  with  fifteen  or 
thirty  per  cent.,  or  even  concentrated,  hydrochloric  acid  for 
six  or  eight  hours  without  undergoing  any  chauge  into 
neurine,  thus  disproving  the  results  obtained  by  Gram. 
E.  Schmidt  has  confirmed  Brieger's  observations  in 
regard  to  the  resistance  of  choline  to  decomposition  by 
acids,  but  he  has  gone  further,  and  has  shown  that  what 
the  action  of  acids  has  failed  to  do  is  readily  accomplished 
through  the  agency  of  bacteria.  He  found  that  choline 
chloride,  when  allowed  to  stand  with  hay  infusion,  or  with 
dilute  blood  for  fourteen  days  at  30°-35°,  it  almost  entirely 
decomposed,  yielding  large  quantities  of  trimethylamine 
and  a  base,  the  platinochloride  of  which  resembles  in  form 
and  solubility  the  double  salt  of  neurine,  and  possesses  a 
similar  physiological  action.  Choline  lactate  in  hay  infu- 
sion developed  an  odor  of  trimethylamine  in  twelve  hours, 
but  at  the  end  of  fourteen  days  a  good  deal  of  choline  was 
still  present.  In  this  case  no  neurine  was  present,  but 
instead  a  homologous  base  was  found,  which  can  be  obtained 
synthetically  by  the  action  of  trimethylamine  on  allyl 
bromide.  According  to  Meyer,  of  Marburg,  this  base 
does  not  possess  the  muscarine-like  action  of  neurine,  but 
resembles  more  closely  pilocarpine. 

Brieger  (I.,  59)  had  unsuccessfully  tried  to  transform 
choline  into  neurine  by  putrefaction.  He  observed  that  the 
choline  decomposed  with  extreme  slowness,  even  when  the 
putrefaction  was  carried  on  at  a  higher  temperature,  yield- 
ing only  trimethylamine.  Wurtz  (1868)  showed  that 
dilute  solutions  of  free  choline  can  be  heated  to  boiling 
without  any  perceptible  decomposition.  Concentrated 
solutions,  however,  decompose  with  the  formation  of  tri- 
methylamine and  glycol,  C2H4(OH)2  (see  page  190).  The 
decomposition  of  choline  was  studied  somewhat  by 
Mauthner  (1873),  who  confirmed  Wurtz's  observation 
that  choline  was  scarcely  decomposed  by  boiling  water,  and 
he  showed  that  when  exposed  to  the  action  of  decomposing 
blood  it  yielded  trimethylamine.  The  results  obtained  by 
K.  Hasebroek  (Zeitsohrift  f.  Physiol.  Ohem.,  12, 151,  1888) 


CHEMISTRY    OF    THE    PTOMAINES.  2-45 

deserve  special  mention  at  this  place.  He  carried  on  the 
putrefaction  of  very  dilute  solutions  of  the  chloride  of 
choline  in  the  presence  of  little  or  no  oxygen  in  Hoppe- 
Seyler  fermentation  flasks.  Sewer  slime,  because  of  its 
strong  fermentative  properties,  was  used  to  induce  the 
putrefaction,  and  calcium  carbonate  was  added  to  neu- 
tralize any  acidity  that  might  develop  during  the  fermen- 
tation. 

The  fermentation,  as  shown  by  the  evolution  of  gases, 
lasted  for  about  three  months.  The  total  quantity  of  gas 
given  off  was  about  one  litre  from  1.17  grammes  choline 
chloride.  The  gases  consisted  almost  entirely  of  carbonic 
acid  and  marsh  gas.  No  hydrogen  was  evolved.  When 
the  fermentation  ceased  the  flask  was  opened  and  several 
cubic  centimetres  of  the  almost  neutral  clear  liquid  were 
injected  under  the  skin  of  a  rabbit  without  producing  the 
least  effect. 

This  liquid  distilled  with  alkali  gave  methylamine 
and  ammonia.  What  is  remarkable  about  this  experiment 
was  the  total  absence  of  the  higher  amines — as,  for  instance, 
trimethylamiue,  which  has  been  observed  so  many  times  as 
a  decomposition-product  of  choline.  The  absence  of  any 
poisonous  base,  as  neurine,  was  probably  largely  connected 
with  the  absence  of  oxygen. 

Free  choline  ordinarily  forms  a  strongly  alkaline  syrup 
which  combines  readily  with  acids  to  form  salts,  most  of 
which  are  deliquescent.  By  oxidation  it  is  converted  into 
betaine  (see  page  249),  and  on  treatment  with  concentrated 
nitric  acid  it  gives  rise  to  muscarine  (see  page  251).  These 
reactions  can  be  represented  by  the  equations  : 


+  H20. 


CH2OH 

CH2 

1 

+  o2  = 

CH2 

N(CH3)3.OH 

N(CH3)3.OH 

Choline. 

Betaine. 

246 


BACTERIAL 

POISONS. 

CH2OH 

1 

CH2OH 

CH2 

1 

+  o  = 

CHOH 

1 

N(CH3)3. 

OH 

N(CH3)3.OH 

Muscarine. 

By  the  action  of  dilute  nitric  acid  choline  is  converted 
into  a  base  the  platinochloride  of  which  is  efflorescent  aud 
corresponds  to  the  formula  (C4H10NT2O3Cl)PtCl4  +  2H20 

(SCHMIEDEBERG  and  HarNACK). 

According  to  Mauti-iner,  choline  resembles  the  caustic 
alkalies  in  its  action.  Although  putrefying  blood  decom- 
poses it  into  trimethylamiue,  yet,  when  present  in  the  pro- 
portion of  1.4  per  cent.,  it  is  said  to  arrest  putrefaction. 
A  1  to  2  per  cent,  solution  is  said  to  dissolve  fibrin  or 
coagulated  albumin  on  boiling. 

The  free  base,  as  well  as  the  carbonate,  is  dimorphous 
and  forms  thin  plates  or  long  needles. 

The  Chloride,  C5H14NO.Cl,  is  easily  soluble  in  water 
and  in  absolute  alcohol  (separation  from  neuridine  hydro- 
chloride). It  crystallizes  over  sulphuric  acid  to  needles 
which  readily  deliquesce  in  the  air. 

The  Platinochloride,  (C5H]4NO.Cl)2PtCl4  (Pt  = 
"81.64  per  cent.),  presents  an  interesting  case  of  trimorph- 
ism.  It  crystallizes  in  monoclinic  plates  (Rinne)  which 
are  easily  soluble  in  water,  insoluble  in  alcohol ;  also  in 
characteristic  superposed  plates,  sometimes  in  the  form  of 
orange-red  flat  prisms  (Brieger).  From  a  warm  saturated 
solution  containing  15  per  cent,  alcohol  it  crystallizes  in 
yellow  regular  octahedra  containing  one  molecule  of  water 
of  crystallization  (Jahns);  from  aqueous  solution  on  slow 
evaporation  it  forms  plates,  clinorhombic  prisms,  or  needles 
(Hoppe-Seyler)  which  are  anhydrous.  When  rapidly 
crystallized  it  forms  prisms  (Hundeshagen,  Jahns, 
Schulze)  ;  and  if  the  solution  is  concentrated  the  prisms 
are  very  thin,  almost  needles.  According  to  Schulze,  it 
sometimes  forms  beautiful  orange-red,  chiefly  six-sided 
plates.     Jahns  maintains  that  the  plates  and  prisms  be- 


CHEMISTRY    OF    THE    PTOMAINES.  247 

long  to  the  same  system  ;  while  Hundeshagen  holds  that 
they  are  distinct.  Instead  of  the  salt  presenting  an  in- 
stance of  trimorphism  as  first  stated  by  Hundeshagen,  it 
would  seem  that  but  two  forms  occur — anhydrous  mono- 
clinic  and  octahedra  with  one  molecule  of  water  of  crystal- 
lization. It  contains  always  more  or  less  water  of  crystal- 
lization which  it  does  not  give  up  completely  over  sulphuric 
acid,  but  only  at  110°  (Brieger).  The  natural  platino- 
chloride  becomes  strongly  electric  on  rubbing,  whereas  the 
synthetic  choline  double  salt  does  not  become  electric.  It 
melts  at  225°  with  effervescence  (Jahns). 

The  Aurochloride,  C5H14NO.Cl.AuCl3  (An  =  44.48 
per  cent.),  is  crystalline  aud  is  difficultly  soluble  in  cold 
water,  but  can  be  recrystallized  from  hot  water  or  from 
boiling  alcohol.  It  forms  prisms,  or  gold-yellow  long 
needles,  which  are  very  easily  soluble  in  hot  water  and 
alcohol  (Lippmann).  It  can  be  separated  from  neuridine 
aurochloride  by  its  solubility  in  water  (Brieger).  Ou 
heating,  the  gold  salt  melts  to  a  brown  liquid  (Schulze) 
and  decomposes  at  264°. 

The  Mercurochloride,  C5H14NO.C1.6HgCl2,  is  ex- 
tremely difficulty  soluble  even  in  hot  water.  On  this 
account  the  mercury  salt  is  very  convenient  for  the  separa- 
tion of  choline  from  accompanying  bases. 

The  Picrate,  C5H14NO.0CcH2(NO2)3,  forms  long,  broad 
needles  which  are  more  easily  soluble  than  neuridine  picrate, 
aud  hence  can  be  separated  by  reorystallization.  It  is  more 
easily  soluble  in  alcohol  than  in  water. 

Physiological  Action  of  Choline. — Choline  was  regarded 
for  a  long  time  as  physiologically  inert,  but  this  belief 
was  set  aside  by  Gaehtgens  (1870),  who  showed  that, 
when  given  in  large  quantity,  it  possessed  a  toxic  action. 
This  observation  of  Gaehtgens  has  siuce  been  con- 
firmed by  Glause  and  Luchsinger,  Brieger,  and 
Boehm.  The  chloride  of  choline  produces  in  animals  the 
same  muscarine-like  symptoms  of  poisoning  as  are  devel- 
oped by  the  vinyl  base  neurine,  the  only  difference  lies  in 
the  intensity  of  the  action.  In  order  to  bring  about  a 
physiological  disturbance,  choline  must  be  given  in  rela- 


248  BACTERIAL    POISONS. 

tiyely  large  doses.  Thus,  Brieger  found  it  necessary  to 
give  about  0.1  gramme  of  choline  chloride  hypodermi- 
cally  to  a  one  kilogramme  rabbit  in  order  to  bring  out  the 
same  effects  as  are  obtained  by  the  injection  of  0.005 
gramme  of  the  neurine  salt.  He  also  found  that  the 
fatal  dose  for  a  one-kilogramme  rabbit  was  about  0.5 
gramme,  which  is  about  ten  times  as  large  as  the  fatal 
dose  of  neurine  chloride.  Boehm  observed  that  doses  of 
0.025-0.1  gramme  produced  in  frogs  general  paralysis, 
which,  in  a  short  time,  leads  to  death  or  recovery;  and 
that  in  its  curara-like  paralyzing  action,  choline  resembles 
artificial  muscarine,  although  the  latter  is  about  five 
hundred  times  stronger.  Atropine,  as  in  the  case  of 
neurine  and  muscarine,  antagonizes  the  action  of  choline. 
Thus,  0.05  gramme  of  the  chloride  produced  in  a  frog  in 
one  hour  diastolic  stoppage  of  the  heart.  This  condition 
was  removed  by  the  injection  of  0  001  gramme  of  atropine, 
the  heart-beat  rising  to  the  normal  in  about  fourteen  min- 
utes ;  0.05  gramme  of  choline  chloride,  given  subcutaue- 
ously  to  a  rabbit  (1250  grammes)  produced  salivation,  which 
lasted  but  a  short  time,  and  did  not  affect  the  heart-beat 
and  respiration  ;  0.10  gramme  was  necessary  to  bring  out 
all  the  symptoms;  0.05  gramme,  given  to  guinea-pigs,  had 
no  effect  whatever. 

Betaine  (Oxyneurine),  C5H13N03. — This  base  has 
been  well  known  for  some  time,  because  of  its  occurrence 
in  the  vegetable  kingdom.  Thus,  it  is  present  in  cotton- 
seed (Boehm,  Bitthausen  and  Weger)  ;  in  beet-root  juice 
(Beta  vulgaris),  and  hence  in  beet-root  molasses  (Schei- 
beer,  1866).  It  occurs  also  in  cattle-turnip  and  Lycium 
barbarum  ;  and  is  found  with  choline  and  another  base  in 
vetch-seeds ;  in  peas  a  base  similar  to  betaine  exists 
(Schulze).  With  choline  it  occurs  in  Scopolia  atropo'ides 
(Siebert).  It  does  not  exist  in  these  substances  as  such, 
but  is  formed  from  a  more  complex  substance  by  the  action 
of  hydrochloric  acid  or  baryta  (Liebreich).  In  this  respect 
it  resembles  choline,  neurine,  and  probably  muscarine. 
Quite  recently,  Lippmann  (1887)  has  obtained  a  lecithin- 


CHEMISTRY    OF    THE    PTOMAINES.  249 

like  body  from  sugar-beet,  which,  on  heating  with  baryta 
gave  oleic  acid,  glycerin,  and  phosphoric  acid  (glycerin- 
phosphoric  acid),  and  betaine.  Betaine,  however,  does  not 
seem  to  be  a  constant  constituent,  inasmuch  as  on  one  occa- 
sion he  obtained  chiefly  choline,  and  little  or  no  betaine. 
These  two  bases  also  occur  together  in  cotton-seed,  and 
this  fact  has  led  Scheibler  to  the  conclusion  that  it  is 
no  mere  chance.  Lecithin,  as  is  well  known,  may  con- 
tain variable  acid  constituents  (oleic,  stearic,  palmitic,  etc.), 
and  reasoning  on  this  fact,  and  on  the  results  of  his  experi- 
ments, Lippmann  has  been  led  to  suppose  that  it  may  also 
contain  different  bases  in  variable  proportions. 

It  has  been  obtained  from  human  urine  (Liebreich, 
1869),  and  from  poisonous  and  non-poisonous  mussel,  but 
not  from  putrid  mussel  (Brieger,  1885,  III.,  76).  The 
method  for  its  separation  from  mussel  is  described  on  page 
255. 

Betaine  may  be  obtained  synthetically  in  several  ways  : 

(1)  by  oxidation  of  choline  with  potassium  permanganate; 

(2)  by  the  action  of  methyl  iodide  on  glycocoll ;  (3)  by 
treating  monochloracetic  acid  with  trimethylamine.  The 
last  two  methods  are  of  value  as  indicating  the  constitution 
of  betaine,  and  the  changes  which  take  place  can  be  repre- 
sented by  the  equations : 

NH2  N(CH3)3I 

CH2         +     3CILI     =     CH2         +       2III. 

C02H  C02H 

Gi.tcocoi.l.  Betaine  Iodide. 


CH2C1 


0O2H 


N(CH3)3C1 

I 

+  N(CH3)3    =    CH2 


C02H. 


MONOCHLOBACETIC   ACID. 

From  the  formulae  of  the  salts  of  betaine  it  is  evident 


250  BACTERIAL    POISONS. 

that  betaine  has  properly  the  composition  C5H13N03,  which 
is  expressed  by  the  structural  formula  : 

N(CH3)3OH 

I 
CH2 

I 

co2n. 

The  free  base  is,  however,  readily  converted  into  the 
anhydride,  C5HnN02,  trimethyl  glycocoll  ;  the  structural 
formula  of  which  is  : 

CH-^(CH3)3 

I  I 

CO  — o. 

Betaine  is  ordinarily  regarded  as  crystallizing  with  one 
molecule  of  water,  and  the  composition  is  expressed  by  the 
formula:  C5HuN02+H20  (=  OH.N(CH3)3.CII2.C62H). 
It  loses  this  water  of  crystallization  by  heating  at  100°,  or 
on  standing  over  sulphuric  acid,  forming  an  anhydride  of 
the  formula  already  given.  Liebreich  claims  that  free 
betaine  possesses  the  formula  C5HuN02,  because  it  yields  a 
compound  having  the  composition  (C5ITnN02)ZnCl2.  The 
free  base  separates  from  alcohol  in  large  crystals  which  deli- 
quesce on  exposure  to  the  air.  As  obtained  by  Brieger 
from  the  hydrochloride  by  treatment  with  moist  silver 
oxide,  it  possessed  a  sweetish  taste  and  neutral  reaction. 
When  distilled  with  potassium  hydrate,  it  yields  trimethyl- 
amine  and  other  bases,  among  which  a  base  of  the  formula 
C8II17N05  occurs  in  the  largest  quantity. 

The  Chloride,  C5H12lNf02. CI,  forms  beautiful  crystals, 
monoclinic  plates,  which  are  permanent  in  the  air,  and  this 
can  be  made  use  of  to  effect  a  separation  from  the  choline 
salt,  which  is  deliquescent.  It  is  insoluble  in  absolute 
alcohol.  This  fact  can  be  made  use  of  in  their  separation 
(Lippmann).  It  can,  moreover,  be  easily  separated  from 
other  bases  by  its  aurochloride,  which  is  easily  soluble.  If 
a  little  potassio-mercuric  iodide  is  added  to  a  solution  of 
the  chloride,   there  forms  a  light-yellow  or  whitish  oily 


CHEMISTRY    OF    THE    PTOMAINES.  251 

precipitate,  which  is  soluble  in  excess,  but  on  rubbing  the 
sides  of  the  tube  with  a  glass  rod  it  reappears  as  yellow 
needles.  This  is  said  to  be  a  characteric  test  (Brieger, 
Schulze,  1891). 

The  Aurochloride,  C6H12N02.Cl.AuCl3  (An  =  43.12 
per  cent.),  forms  magnificent  cholesterin-like  plates,  and  is 
easily  soluble  (Brieger).  The  aurochloride  from  sugar- 
beet  is  said  to  crystallize  in  needles  or  plates,  and  to  be 
difficultly  soluble  in  cold  water  (Scheibler,  Lippmann). 
The  double  salt  of  the  ptomaine  melts  at  209°,  and  in  this 
it  coincides  with  that  obtained  from  beet- sugar,  as  well  as 
with  that  of  the  synthetically  prepared  base  (Brieger). 
The  platinochloride  is  yellow  and  crystalline. 

Betaine  is  not  poisonous. 

Muscarine,  C6H15N03  =  C5H131ST02  +  H20,  the  well- 
known  toxic  principle  which  Schmiedeberg  obtained 
from  poisonous  mushroom  (Agaricus  muscarius),  has  been 
obtained  also  by  Brieger  in  1885  (L,  48)  from  haddock 
which  had  been  allowed  to  decompose  for  five  days.  The 
process  by  which  its  isolation  was  effected  is  described  on 
page  258.  This  base  is  specially  interesting,  because  of  the 
relation  it  bears  to  choline,  for  Schmiedeberg  has  shown 
that  it  is  formed  when  choline,  or,  better  still,  the  platino- 
chloride, is  oxidized  by  concentrated  nitric  acid.  It  is 
barely  possible  that  Brieger's  base  is  distinct  from 
Schmiedeberg's  ;  nevertheless,  it  closely  resembles  it  and 
apparently  is  identical. 

The  Chloride,  C5H14N02.C1,  is  obtained  on  the  decom- 
position of  the  platinochloride  with  hydrogen  sulphide,  as 
a  syrupy  residue,  which,  under  the  desiccator,  shows  a 
tendency  to  gradually  crystallize.  It  is  deliquescent 
(Harnack)! 

The  Platinochloride,  (C5H14N02.Cl)2PtCl4  (Pt  = 
30.08  per  cent.),  forms  as  a  crystalline  deposit  of  octahedra, 
which  are  difficultly  soluble  in  water.  They  lose  their 
water  of  crystallization  (2H20)  only  by  means  of  strong 
heating. 

The   Aurochloride,    C6HuN02.C1.AuC13,  crystallizes 


252 


BACTERIAL    POISONS 


in  needles,  and  is  difficultly  soluble  in  water,  more  so  than 
the  choline  double  salt  (Harnack). 

Physiological  Action. — Small  doses  of  this  ptomaine 
induce  in  frogs  total  paralysis,  with  stoppage  of  the  heart 
in  diastole,  and  this  action  is  antagonized  by  subsequent 
injection  of  atropine,  as  well  as  in  the  case  of  previously 
atropinized  frogs.  Very  small  doses  produce  in  rabbits 
profuse  salivation  and  lachrymation,  contraction  of  the 
pupil,  profuse  diarrhoea,  and  passage  of  urine  and  semen ; 
finally,  the  animal  dies  in  convulsions,  which,  however,  are 
only  of  short  duration. 

Constitution  of  the  Members  of  the  Choline 
Group. — The  structure  of  choline  was  clearly  demon- 
strated by  Wurtz,  who  accomplished  the  synthesis  of  this 
base  by  treatment  of  ethylene  chlorhydrine  with  trimethyl- 
amine.  This  same  method  can  be  applied  to  the  synthe-is 
of  betaine  aud  neurine  by  using  monochloracetic  acid  and 
vinylbromide  instead  of  ethylene  chlorhydrine.  The  struc- 
tural formulas  which  can  be  deduced  from  these  reactions 
are  as  follows  : 


CII2OH            CTI2 

1                       II 

C02H 

CII2OH 

1 

CH2                 CH 

1                       1 

CH2  . 

1 

CHOH 

1 

N(CH3)3.OH    N(CH3)3.OH 

Choline.                        Neurine. 

N(CH3)3.OH 

Betaine. 

N(CH3)3.OH 

Muscarine. 

The  formulas  of  betaine  and  muscarine  are  ordinarily  given 
as  the  anhydrides,  but  there  can  be  no  doubt  that  the  free 
bases  possess  the  structure  indicated  above.  All  these  bases, 
since  they  can  be  prepared  from  choline,  may  also  be  con- 
sidered as  oxidation-products  of  trimethyl-ethyl-ammonium 
hydrate  : 

CH, 


CH0 


N(CII3)3.OH. 


CHEMISTRY    OF    THE    PTOMAINES.  253 

Mydatoxine,  C6H13N02. — This  base  was  obtained  by 
Brieger  iu  1886  (III.,  25,  32)  from  several  hundred 
pounds  of  human  internal  organs  which  were  allowed  to 
stand  in  closed  but  spacious  wooden  barrels  for  four  months, 
at  a  temperature  varying  from  — 9°  to  +5°.  He  obtained 
much  larger  quantities  of  it,  however,  from  horseflesh  which 
had  putrefied  under  the  same  conditions.  In  the  process 
of  extraction  it  is  found  in  the  mercuric  chloride  precipitate 
together  with  cadaverine,  putrescine,  and  another  base, 
C7H17N02.  It  can  be  isolated  from  this  mixture  by  recrys- 
tallizing  the  mercury  salts,  which  removes  the  cadaverine, 
because  of  its  difficult  solubility  in  water,  and  decomposing 
the  soluble  mercury  salts  by  hydrogen  sulphide.  The 
filtrate  freed  from  mercury  is  now  evaporated  to  dryness 
and  the  residue  repeatedly  extracted  with  absolute  alcohol, 
in  order  to  remove  putrescine  hydrochloride,  which  is 
insoluble.  The  alcoholic  solution,  after  standing  some  time 
to  permit  complete  separation  of  any  dissolved  putrescine, 
is  then  evaporated  to  dryness  and  taken  up  with  water. 
This  solution  gives,  on  the  addition  of  gold  chloride,  a  pre- 
cipitate of  the  aurochloride  of  the  base  C7H17N02.  The 
filtrate  from  this  precipitate,  containing  the  mydatoxine,  is 
treated  with  hydrogen  sulphide  to  remove  the  gold,  and 
then  evaporated  to  dryness.  The  colorless,  syrupy  hydro- 
chloride thus  obtained  forms  with  platinum  chloride  a 
double  salt  which  is  readily  soluble  in  water,  and  can  be 
purified  by  repeated  recrystallizations  from  absolute  alcohol 
containing  some  hydrochloric  acid. 

The  name  mydatoxine  is  derived  from  pvd&a,  to  putrefy. 
The  free  base  is  obtained  from  the  hydrochloride  by  treat- 
ment with  moist,  freshly  precipitated  silver  oxide,  as  a 
strongly  alkaline  syrup,  which  solidifies  in  vacuo  to  plates. 
It  is  insoluble  in  alcohol,  ether,  etc.  It  does  not  distil 
without  decomposition.  It  is  isomeric  with  the  base, 
06H13NO2,  obtained  by  Brieger  in  1888  from  tetanus 
cultures. 

The  Hydrochloride,  C6H13TST02.HC1,  is  a  colorless, 
deliquescent  syrup  which  does  not  form  any  double  salt 
with  gold  chloride.     With  platinum  chloride  it  gives  an 

12 


254  BACTEKIAL    POISONS. 

easily  soluble  salt.  Otherwise  it  combiues  only  with  phos- 
phoraolybdic  acid,  with  which  it  forms  cubes.  Ferric 
chloride  aud  potassium  ferricyanide  yield,  after  a  time, 
Berlin-blue.     It  is  readily  soluble  iu  alcohol. 

The  Platinochloride,  (C6H13N02.IICl)2PtCl4>  (Pt  = 
29.00  per  cent.),  melts  at  193°,  with  decomposition.  It 
crystallizes  iu  plates  which  are  extremely  soluble  in  water. 
It  can  be  readily  recrystallized  from  absolute  alcohol  acidu- 
lated with  hydrochloric  acid.  The  mercury  salt  is  readily 
soluble  in  water. 

The  exact  formula  of  this  base,  of  mytilotoxine,  and 
some  other  bases,  cannot  be  considered  to  be  permanently 
settled,  inasmuch  as  the  formula  of  the  hydrochloride, 
06H13NO2.IICl,  as  deduced  from  the  analysis  of  the 
platinum  double  salt,  may  equally  apply  to  the  base 
C6HuN02.OH  as  to  the  base  C;ilI3N02.  If  the  first 
formula  is  correct,  then  mydatoxiue  is  a  homologue  of 
betaine,  and  its  structure  would  be  expressed  by  (1). 

(i)  (2) 

COJI  CS° 

I  I  xn 

CH2  CII 

I  II 

GIL  OH 

I  I 

N(CII3)3OH  N(CII3)3OII. 

The  second  formula  would  seem  to  correspond  to  an  un- 
saturated aldehyde  of  the  choline  group  and  its  structure 
may  be  indicated  by  (2). 

This  ptomaine,  although  it  possesses  toxic  properties,  is 
not,  however,  a  strong  poison.  Its  action  is  the  same  as  that 
of  the  base  07IT17NO2  (see  page  262),  with  which  it  is  associ- 
ated, except  that  the  symptoms  of  poisoning  develop  slower, 
so  that  the  death  of  a  guinea-pig  does  not  take  place  for 
about  twelve  hours.  White  mice  are  very  susceptible  to  the 
action  of  these  two  poisons.  A  short  time  after  the  injec- 
tion of  even  small  doses  they  are  taken  with  convulsions 


CHEMISTRY    OF    THE    PTOMAINES.  255 

which  come  on  in  paroxysms.  The  eyeballs  roll  upward. 
Lachrymation,  diarrhoea,  and  dyspnoea  come  on,  and  the 
mice  die  within  a  short  time. 

A  Base  (?),  C6H13lSr02,  an  isomer  of  the  preceding,  was 
obtained  by  Brieger  in  1888  from  tetanus  cultures.  It 
is  not  poisonous — distinction  from  mydatoxine.  It  proba- 
bly is  an  amido-acid.  The  platinochloride  crystallizes  in 
plates,  is  easily  soluble  in  water  and  in  alcohol,  and  melts 
at  11*7°  with  decomposition  (see  page  267). 

Mytilotoxixe,  C6H15]Sr02,  is  the  specific  poison  of  toxic 
mussel  (Mytilus  edulis),  from  which  it  was  obtained  by 
Brieger  in  1885  (III.,  76).  .This  poison  is  formed  during 
the  life  of  the  animal  under  certain  conditions  which  have 
been  thoroughly  studied  by  Schmidtmann,  Virchowt,  and 
others  (see  p.  40).  Brieger  obtained  the  poison  by  extract- 
ing toxic  mussel  with  acidulous  water,  and  evaporating  this 
solution  to  a  syrupy  consistency.  The  residue  was  thor- 
oughly extracted  with  alcohol,  and  this  solution  was  treated 
with  lead  acetate,  in  order  to  remove  mucilaginous  sub- 
stances. The  filtrate  was  then  evaporated,  and  the  residue 
extracted  with  alcohol.  Any  lead  that  had  dissolved  was 
removed  by  hydrogen  sulphide.  The  alcohol  was  expelled, 
and  the  resulting  syrup  was  taken  up  with  water  and 
decolored  by  boiling  with  animal  charcoal.  The  clear  solu- 
tion was  now  neutralized  with  sodium  carbonate,  acidulated 
with  nitric  acid,  and  precipitated  with  phosphomolybdic 
acid.  The  precipitate  was  decomposed  by  warming  with 
neutral  lead  acetate,  and  the  resulting  nitrate,  after  the 
removal  of  the  lead  by  hydrogen  sulphide,  was  acidulated 
with  hydrochloric  acid  and  evaporated  to  dryness.  The 
residue  was  extracted  with  absolute  alcohol,  whereby 
betaine,  on  account  of  its  insolubility,  is  removed,  and  the 
alcoholic  solution  was  precipitated  by  alcoholic  mercuric 
chloride.  The  mercury  precipitate  is  repeatedly  recrystal- 
lized  from  water,  and  the  poison  is  obtained  as  an  easily 
soluble  double  salt. 

The  free  base  as  obtained  by  the  addition  of  alkali  to 


256  BACTERIAL    POISONS. 

the  hydrochloride  possesses  a  disagreeable  odor  which  dis- 
appears on  exposure  to  air,  and  the  substance  ceases  to  pos- 
sess poisonous  properties.  Brieger  has  proposed  the 
application  of  this  test  for  the  recognition  of  poisonous 
mussel ;  on  treatment  of  these  with  alkali  the  characteristic 
odor  is  developed.  Mytilotoxiue  is  also  destroyed  on  dis- 
tillation with  potassium  hydrate  and  in  the  distillate  there 
is  found  an  aromatic  non-poisonous  product  and  trimethyl- 
amine.  The  free  base,  therefore,  does  not  exist  by  itself 
for  any  length  of  time,  but  soon  becomes  converted  into  an 
inert  substance.  H.  Salkowski  has  also  shown  that  it  is 
destroyed  on  boiling  with  potassium  carbonate,  whereas 
its  hydrochloric  acid  solution  can  be  evaporated  to  dry- 
ness and  heated  to  110°  without  destroying  its  poisonous 
property. 

The  Hydrochloride,  C6H15NO2.H01,  prepared  from 
the  aurochloride,  crystallizes  in  tetrahedra.  It  is  extremely 
poisonous  and  according  to  Brieger  produces  exactly  the 
same  symptoms  which  have  beeu  observed  by  Schmidt- 
mann  in  persons  who  have  partaken  of  poisonous  mussels 
(see  page  38).  On  standing,  however,  the  pure  hydro- 
chloride gradually  becomes  dark  and  decomposes  with  loss 
of  its  poisonous  property — a  change  corresponding  to  that 
which  tetanine  undergoes  (p.  267).  The  gold  salt  is  better 
adapted  for  preservation.  The  ordinary  alkaloidal  reagents 
produce  in  its  solutions,  if  at  all,  only  oily  precipitates. 

As  stated  uuder  mydatoxine,  the  formula  of  the  hydro- 
chloride, C6II15N02.LlC1,  is  applicable  to  either  one  of  two 
bases,  C6H16N02.OH  or  C6H15N02.  The  base  correspond- 
ing to  the  first  formula  is  evidently  a  homologue  of  mus- 
carine, and  should  possess  a  similar  physiological  action. 
As  a  matter  of  fact,  mytilotoxine  does  resemble  muscarine 
somewhat  in  its  action,  and  its  occurrence  together  with 
betaine  would  seem  to  make  it  a  decomposition-product  of 
lecithin,  in  which  case  this  base  must  be  looked  upon  as  a 
member  of  the  choline  group.  It  is  interesting  to  know 
thata  compound  corresponding  to  the  formula  C6H16N02.OH 
has  been  known  for  some  time,  and  was  prepared  by  Han- 
riot   in    a  manner  analogous  to   Wurtz's  synthesis  of 


CHEMISTEY    OF    THE    PTOMAINES.  257 

choline,  by  treating  glycerin  monochlorhydrine  with  tri- 
methylamine.  This  base,  trimethyl-glyceryl-ammonium 
hydrate,  has  this  structure : 

CH2OH 

I 
CHOH 

I 
CH2 

I 
N(CH3)3OH. 

It  would  seem  that  Hanriot's  base  might  possibly  be 
identical  with  mytilotoxine,  but  a  careful  comparison  made 
by  Brieger  showed  that  it  possesses  no  physiological  actiou 
aud  that  its  chemical  reactions  are  entirely  different. 

Mytilotoxine  would,  therefore,  seem  to  possess  the  for- 
mula, C6H15N02,  as  originally  given  it  by  Brieger.  From 
the  fact  that  on  distillation  with  potassium  hydrate  it  yields 
trimethylamine,  it  follows  that  mytilotoxine  is  a  quarter- 
nary  base.  He  is  inclined  to  regard  it  as  a  methyl  deriva- 
tive of  betaine,  which  is  so  common  in  mussels,  aud  repre- 
sents it  by  formula  No.  1. 

(1)  (2) 

C02H  CH,OH 

I  I 

CH.CH3  CH.CH3 

I  I 

N(CH3)3.OH  N(CH3)3.OH 

No.  1,  however,  is  C6H15N03,  instead  of  C6H15N02,  as 
above.  The  formula  No.  2,  C6H17N02,  would  represent 
a  derivative  of  choline  or  muscarine,  with  only  a  slightly 
higher  percentage  of  hydrogen. 

The  Aurochloride,  C6HJ5N02.HCl.AuCl3(Au  =  41.66 
per  cent.),  crystallizes  in  cubes.     Its  melting-point  is  182°. 

It  is  well  to  observe  that  Brieger  has  been  unable  to 
obtain  this  base  from  mussels  that  were  allowed  to  putrefy 
for  sixteen  days. 

Physiological  Action. — According  to  Brieger,  mytilo- 
toxine produces  all  the  characteristic  effects  seen  in  mussel 


258  BACTERIAL    POISONS. 

poisoning,  and  it  is,  therefore,  a  strong  paralysis-producing 
poison,  and  resembles  curara  in  its  action.  This  action  is 
explainable  now  that  Glause  and  Luchsinger  have 
shown  that  all  trimethyl-ammonium  bases  have  a  musca- 
rine-like  action.  For  the  symptoms  induced  by  poisonous 
mussel  see  page  38. 

Gadinine,  C7H17N02,  was  found  in  haddock  (1885) 
which  was  allowed  to  decompose  in  open  iron  vessels  for 
five  days  during  summer.  Brieger  has  also  obtained  it 
from  cultures  of  the  bacteria  of  human  feces  on  gelatin. 
The  decomposing  mass  was  thoroughly  stirred  every  day 
in  order  to  bring  it  into  contact  with  atmospheric  oxygen 
(Brieger,  I.,  49).  It  was  then  treated  with  water,  and 
hydrochloric  acid  was  added  to  acid  reaction,  and  after  being 
warmed  the  mixture  was  filtered  and  the  nitrate  concen- 
trated on  the  water-bath  to  a  syrupy  consistency.  This 
syrupy  residue  was  extracted  with  water,  and  the  aqueous 
solution  was  precipitated  with  a  solution  of  mercuric  chlo- 
ride. The  mercuric  chloride  precipitate  contained  a  base, 
the  quantity  of  which,  however,  was  insufficient  for  a  com- 
plete analysis  (see  page  272).  The  mercuric  chloride  filtrate, 
after  the  removal  of  the  mercury  by  hydrogen  sulphide, 
was  evaporated  to  a  syrup,  and  this  was  then  repeatedly 
extracted  with  alcohol.  The  alcoholic  solution  thus  ob- 
tained contained  neuridine,  a  base  of  the  same  composition 
as  ethylenediamine,  muscarine,  gadinine,  and  triethylamine. 
These  bases  were  separated  in  the  following  manner :  The 
alcoholic  solution  gave  with  platinum  chloride  a  precipitate 
of  neuridine.  The  filtrate  from  this  platinum  precipitate 
was  heated  on  the  water-bath  to  expel  the  alcohol,  and  then 
the  platinum  was  removed  by  hydrogen  sulphide.  The 
aqueous  filtrate  was  concentrated  to  a  small  volume  which, 
on  addition  of  platinum  chloride,  gave  a  precipitate  of  the 
isomer  of  ethylenediamine.  The  mother-liquor  from  this 
precipitate  was  concentrated  on  a  water-bath,  and  on  cool- 
ing the  platinochloride  of  muscarine  crystallized  out.  From 
the  mother-liquor  of  this  precipitate  on  standing  in  a  des- 
iccator, the  gadinine  double  salt  crystallized.     The  mother- 


CHEMISTRY    OF    THE    PTOMAINES.  259 

liquor  from  the  gadinine  platiuochloride  was  treated  with 
hydrogen  sulphide  to  remove  the  platinum,  aud  the  aque- 
ous ^-filtrate  on  distillation  with  potassium  hydrate  gave 
triethylamiue. 

Gadinine  (from  Gadus  callarias,  haddock)  in  small  doses 
does  not  appear  to  be  poisonous ;  larger  doses  (0.5-1  gramme) 
are  decidedly  toxic  and  may  kill  guinea-pigs.  The  formula 
of  the  free  base  as  deduced  from  the  analysis  of  the  platiuo- 
chloride may  be  either  C7II17N02  or  C7H18N02  OH. 

The  Hydrochloride,  C7H17N02.HC1,  as  obtained  by 
the  decomposition  of  the  platiuochloride  with  hydrogen 
sulphide,  crystallizes  under  the  desiccator  in  thick,  colorless 
needles,  which  are  easily  soluble  in  water ;  insoluble  in 
alcohol.  It  forms  no  combination  with  gold  chloride,  but 
does  give  crystalline  precipitates  with  phosphomolybdic 
acid,  phosphotungstic  acid,  and  picric  acid. 

The  Platiuochloride,  (C7H17N02.HCl).2PtCl1  (Pt  = 
27.68  per  cent.),  is  at  first  quite  soluble,  and  on  standing  over 
a  desiccator  it  crystallizes  in  golden-yellow  plates,  which, 
when  once  formed,  are  again  difficultly  soluble  in  water. 
It  can  be  recrystallized  from  hot  water.     It  melts  at  214°. 

Typhotoxlne,  C7H17N02. — This  base  was  named  thus 
by  Brieger  in  1885  (III.,  86),  and  is  regarded  by  him  as 
the  specific  toxic  product  of  the  activity  of  Koch-Eberth's 
typhoid  bacillus.  It  is,  however,  probable  that,  as  in  the 
case  of  tetanus,  there  are  basic  and  other  products  formed. 
He  obtained  it  by  cultivating  the  bacillus  on  beef-broth  for 
eight  to  fourteen  days  at  the  temperature  37.5-38°.  The 
nature  of  the  soil  on  which  it  grows  has  a  great  deal  to  do 
with  the  formation  of  the  poison.  An  especially  important 
factor  is  the  temperature  :  for  Brieger  has  observed  that 
no  poison  was  produced  in  one  case  where  the  temperature 
remained  by  accident  at  39°  for  twenty-four  hours.  In 
such  cases  creatine  is  present  in  quantity,  whereas  otherwise 
the  reverse  is  the  rule. 

In  the  process  of  extraction  it  occurs  in  the  mercuric 
chloride  precipitate,  and  from  this  it  is  obtained,  after  the 
removal  of  the  mercury  by  hydrogen  sulphide,  as  an  easily 


260  BACTEKIAL    POISONS. 

deliquescent  hydrochloride.  This  for  the  purpose  of  puri- 
fication is  converted  into  the  difficultly  soluble  aurochloride. 

Typhotoxine  is  isomeric  with  gadiniue  and  the  compound 
C7H17N02,  which  Brieger  obtained  from  putrefying  horse- 
flesh. In  its  properties  it  is,  however,  very  different. 
Thus,  the  free  base  is  strongly  alkaline  and  its  hydrochloride 
yields  a  difficultly  soluble  picrate.  On  the  other  hand,  the 
isomer  from  horseflesh  possesses  a  slightly  acid  reaction, 
and  does  not  form  a  picrate.  Again,  typhotoxine  gives 
with  Ehrlich's  reagent  (sulpho-diazobenzole)  an  imme- 
diate yellow  color,  which  disappears  upon  the  addition  of 
alkali,  whereas  the  isomer  does  not  give  this  reaction. 
Furthermore,  the  two  bases  differ  in  their  physiological 
action  and  in  their  behavior  to  alkaloidal  reagents  (see 
Table  I.).  Their  aurochlorides,  however,  possess  the  same 
melting-point. 

The  Hydrochloride  is  readily  deliquescent,  and  unites 
with  platinum  chloride  to  form  an  easily  soluble  double 
salt  crystallizing  in  needles. 

The  Aurochloride,  07H17NO2.HCl.  AuC13  (Au  =  40.46 
per  cent.),  is  difficultly  soluble,  and  crystallizes  in  prisms, 
which  melt  at  176°.  In  its  melting-point  and  solubility 
(197°,  Brieger,  Arch.  f.  pathol.  Anat.,  115,  489)  it 
agrees  with  its  isomer  from  horseflesh.  From  some  of  his 
first  experiments  in  the  cultivation  of  the  typhoid  bacillus, 
Brieger  (II.,  69)  obtained  a  basic  product  differing  in 
some  of  its  characters  from  typhotoxine.  Its  aurochloride, 
on  analysis,  gave  41.91  and  41.97  per  cent,  of  Au,  16.06 
per  cent,  of  C,  and  3.66  per  cent,  of  H. ;  while  typho- 
toxine aurochloride  gave  40.78  per  cent.  Au,  17.38  per 
cent.  C,  and  3.85  per  cent.  H.  For  a  comparison  of  the 
reaction  of  these  two  substances  see  Table  I. 

In  its  physiological  action,  typhotoxine  differs  from  its 
isomer  (page  262)  in  that  the  latter  produces  symptoms 
with  well-marked  convulsions,  whilst  the  former  throws 
the  animal  into  more  of  a  paralytic  or  lethargic  condition. 
The  action  of  this  base  has  been  studied  only  on  mice  and 
guinea-pigs.  It  produces  at  first  slight  salivation  with 
increased  respiration;  the  animals  lose  control  over  the 


CHEMISTRY    OF    THE    PTOMAINES.  261 

muscles  of  the  trunk  and  extremities,  and  fall  down  help- 
less upon  their  sides.  The  pupils  become  strongly  dilated, 
and  cease  to  react  to  light ;  the  salivation  becomes  more 
profuse ;  the  rate  of  heart-beat  and  of  respiration  gradually 
decreases,  and  death  follows  in  from  one  to  two  days. 
Throughout  the  course  of  these  symptoms  the  animals 
have  frequent  diarrhceic  evacuations,  but  at  no  time  are 
convulsions  present.  On  post-mortem,  the  heart  is  found 
to  be  in  systole,  the  lungs  are  strongly  hypersemic,  the 
other  internal  organs  pale,  the  intestines  firmly  contracted, 
and  their  walls  pale. 

A  Base(?),  C7H17N02,  was  obtained  by  Brieger  in  1886 
(III.,  28)  on  working  over  about  one  hundred  pounds  of 
horseflesh  which  had  been  allowed  to  undergo  slow  putre- 
faction with  limited  access  of  air  and  at  a  low  temperature 
( — 9°  to  +  5°)  for  four  months.  It  occurs  in  the  mercuric 
chloride  precipitate  together  with  cadaverine,  putrescine, 
and  mydatoxine,  and  from  these  bases  it  can  be  separated 
and  isolated  according  to  the  method  on  page  233. 

A  similar,  if  not  identical  substance,  having  the  com- 
position C7H17N02,  was  obtained  by  Baginsky  and  Stadt- 
hagen  (1890)  from  cultures  on  horseflesh,  ten  days  at  85°, 
of  a  bacillus,  closely  allied  to  Finkler-Prior's,  and  iso- 
lated from  stools  of  cholera  infantum.  The  gold  salt  in 
crystalline  form  and  properties  is  the  same  as  Brieger's, 
except  that  it  possesses  a  somewhat  higher  melting-point. 

The  free  substance  possesses,  even  after  most  careful 
purification,  a  slightly  acid  reaction.  This  acidity  is 
removed  from  even  a  large  quantity  of  the  substance  by 
the  addition  of  a  drop  of  alkali.  On  account  of  the  acid 
character  of  the  free  substance,  Brieger  does  not  consider 
it  to  be  a  base  (a  ptomaine).  It  differs,  however,  from  the 
amido-acids  in  its  poisonous  character ;  in  the  fact  that, 
unlike  an  acid,  it  does  not  unite  with  bases  to  form  salts ; 
and  in  not  giving  the  characteristic  red  coloration  (Hof- 
meister's  reaction  for  the  amido-acids)  with  ferric  chloride. 
Whatever  the  true  nature  of  this  substance  may  be,  it 
nevertheless,  in  its  other  properties,  behaves  like  a  base. 

12* 


262  BACTERIAL    P0I30XS. 

Thus,  it  forms  simple  as  well  as  double  salts.  On  boiling 
with  copper  acetate,  it  gives  amorphous  floccules.  Under 
the  desiccator  it  solidifies  into  plates  which  deliquesce  on 
exposure  to  the  air.  It  does  not  combine  either  with  silver 
oxide  or  with  cupric  hydrate.  On  dry  distillation  it  yields 
a  distillate  possessing  a  strong  acid  reaction  and  a  peculiar 
odor.  The  distillate  does  not  give  any  precipitate  with 
platinum  chloride,  or  with  gold  chloride,  nor  does  it  react 
with  copper  acetate.  With  phosphomolybdic  acid,  how- 
ever, it  forms  an  amorphous  mass  :  with  ferric  chloride  and 
potassium  ferricyanide  it  yields  an  immediate  precipitate  of 
Berlin-blue,  whereas  the  original  substance  does  not  give 
any  blue  coloration. 

The  Hydrochloride,  •C7HI7X02.HC1,  crystallizes  in 
fine  needles  which  are  insoluble  in  absolute  alcohol.  When 
its  aqueous  solution  is  treated  with  freshly  precipitated 
silver  oxide,  the  resulting  filtrate  contains  some  silver  oxide 
in  solution,  from  which  it  can  be  removed  by  hydrogen 
sulphide;  thus  differing  from  an  ammoniacal  silver  solu- 
tion, which  gives  no  precipitate  on  treatment  with  hydrogen 
sulphide.  In  this  respect  it  resembles  Salkowski's  base. 
page  231.    For  reactions  of  the  hydrochloride,  see  Table  I. 

The  Aurochloride,  C7HlrN02.HCl.AuCl3,  forms 
plates  which  are  difficultly  soluble  in  water,  and  melt  at 
176° — the  melting  point  of  the  gold  salt  of  typhotoxine. 
It  is  dimorphous,  since  sometimes  it  is  also  obtained  in 
needles  which  can  be  changed  into  plates. 

It  does  not  form  a  picrate,  nor  does  it  give  a  reaction 
with  sulpho-diazobenzole. 

Physiological  Action. — This  substance,  when  injected 
into  frogs,  produces  a  curara-like  action.  A  few  minutes 
after  the  injection  the  animal  falls  into  a  condition  of 
paralysis,  and,  although  it  can  still  react  toward  reflexes,  it 
cannot  move  from  its  place.  At  times  fibrillary  twitchings 
pass  over  the  body.  The  pupils  dilate,  the  heart-action 
becomes  gradually  weaker,  and  finally,  after  several  hours 
the  animal  dies,  with  the  heart  in  diastole.  Doses  of  0.05  to 
0.3  gramme  of  the  hydrochloride,  injected  into  guinea-pigs, 
produce  in  a  short  time  a  slight  tremor,  gradual  increase  in 


CHEMISTRY    OF    THE    PTOMAINES.  263 

respiration,  and  slight  moistening  of  the  lower  lip.  The 
pupils  at  first  contract,  then  dilate  ad  maximum,  and  become 
reactionless.  The  temperature  remains  at  first  normal ; 
chills  of  short  duration  follow  in  rapid  succession.  The 
animal  squats  on  the  ground  with  its  snout  pressing  against 
the  floor  in  exactly  similar  manner  as  is  caused  by  the 
mussel  poison.  Violent  clonic  convulsions  follow  in  con- 
tinually shorter  intervals,  and  at  the  same  time  lachryma- 
tion  and  salivation  become  profuse,  but  not  as  excessive 
as  in  the  case  of  the  muscariue-like  ptomaines.  The  tem- 
perature sinks  with  the  decrease  in  the  rate  of  respiration, 
the  ears  previously  gorged  become  pale  and  cold,  and  the 
heart-action  becomes  irregular  and  less  frequent  than  before. 
General  paralysis  sets  in,  but  the  head  still- moves  upward 
and  backward.  External  stimuli  induce  violent  clonic 
convulsions,  the  animal  repeats  frequently  choking  move- 
ments, and  at  the  same  time  yields  large  quantities  of 
saliva;  finally,  it  falls  upon  its  side  completely  paralyzed, 
and  dies.  The  heart  stops  in  diastole,  the  intestines  are 
pale  and  strongly  contracted,  and  the  bladder  is  empty  and 
likewise  contracted. 

Morrhuic  Acid,  C9Hlsy03,  was  obtained  by  Gautier 
and  MOURC4UES  (1888)  from  brown  cod-liver  oil,  together 
with  six  bases,  already  described — namely,  butylamiue, 
amylamine,  hexylamine,  dihydrolutidine,  aselline  and  mor- 
rhuiue.  These  bases  constitute  about  0.2  per  cent,  of  the 
oil.  The  discoverers  regard  them  as  true  leucomaines, 
dissolved  from  the  hepatic  cells  by  the  oil.  It  is  more 
probable,  however,  that  these  compounds  are  the  products 
of  initial  decomposition,  and  for  that  reason  they  are  de- 
scribed under  the  head  of  ptomaines.  This  compound  is 
relatively  abundant,  and  is  basic  as  well  as  acid  in  charac- 
ter. It  is  resinous  in  appearance,  and  can  be  crystallized 
in  flattened  prisms,  or  large  lance-shaped  plates.  When 
recently  precipitated  it  is  oleaginous,  viscous,  then  gradually 
hardens.  It  possesses  a  disagreeable  aromatic  odor  re- 
sembling that  of  the  sea-weeds,  upon  which  the  fish  feed. 
According  to  the   discoverers   its   probable   source  is  the 


264  BACTERIAL    POISONS. 

lecithin  derived  thus  from  these  weeds.  It  is  soluble  in 
alcohol,  and  but  slightly  in  ether.  It  reddens  turmeric, 
decomposes  carbonates  aud  with  acids  forms  salts  which 
precipitate  lead  acetate  and  silver  nitrate,  but  not  copper 
acetate,  even  on  warming. 

The  hydrochloride  is  crystalline,  and  is  partially  disso- 
ciated by  excess  of  water.  The  platinum  salt  is  soluble, 
and  crystallizes  in  very  small  cross-shaped  prismatic 
needles.  The  gold  salt  is  amorphous  and  is  readily  altered 
on  heating. 

The  properties  of  this  compound  show  that  it  is  of  a 
pyridine  nature,  and  inasmuch  as  it  does  not  give  a  pre- 
cipitate with  copper  acetate,  it  would  appear  that  the  carb- 
oxyl  is  not  directly  united  to  the  pyridine  nucleus.  This 
does  not  necessarily  follow  now  that  we  know  that  some 
amido-acids  exists  which  do  not  give  a  reaction  with  copper 
acetate  (see  page  231).  Its  pyridine  nature  is  further- 
more shown  on  distillation  with  lime.  An  oily  alkaline 
base  is  thus  obtained  which  forms  an  iodomethylate,  and 
this  with  potassium  hydrate  yields  quite  an  intense  red 
color,  resembling  lees  (De  Coninck's  reaction).  On 
oxidation  with  permanganate  of  potassium  it  yields  a  mono- 
basic acid.  According  to  Gautier  and  Mourgtjes  the 
compound  is  probably  identical  with  De  Jungh's  gaduine, 
and  they  ascribed  to  it  the  following  constitution,  which, 
it  should  be  said,  lacks  full  confirmation  : 

H 
C 
<?  \ 
HC       COH 

I         II 
H2C        C— C3H6.CO.,H. 

\   / 

N 

H 

Compare  with  tyrosin,  C9HuN03  (page  197). 


CHEMISTRY    OF    THE    PTOMAINES.  265 

A  Base,  C5H12N204,  was  obtained  by  Pouchet  (1884) 
from  the  residual  liquors  resulting  from  an  industrial  treat- 
ment of  debris  of  bones,  flesh,  and  waste  of  all  kinds,  with 
dilute  sulphuric  acid.  It  is  accompanied  by  another  base, 
C7H18N206,  from  which  it  can  be  separated  by  treatment 
with  alcohol.  The  base  itself  forms  tufts  of  delicate  needles 
which  alter  or  decompose  less  easily  than  the  accompanying 
base.  The  platiuochloride,  (C5Hl2N2O4.HCl)2Pt014,  forms 
a  dull  yellow  powder,  somewhat  soluble  in  strong  alcohol, 
but  insoluble  in  ether.  The  platinochloride  (C7H18N206. 
HCl)2PtCl4  is  insoluble  in  ether. 

The  hydrochlorides  of  these  bases  form  silky  needles 
which  are  altered  by  excess  of  hydrochloric  acid  and  by 
exposure  to  air.  Pouchet  considers  them  to  be  closely 
allied  to  the  oxy-betaines.  The  general  alkaloidal  reagents 
precipitate  these  bases ;  the  phosphomolybdic  precipitate, 
on  addition  of  ammonia,  gives  a  blue  tint.  Both  bases  are 
toxic,  and  exert  a  paralyzing  action  upon  the  reflex  move- 
ments. 

The  method  employed  by  Pouchet  for  their  isolation 
was  to  precipitate  them  as  tauuates.  The  precipitate  was 
decomposed  by  lead  hydrate  in  the  presence  of  strong 
alcohol,  the  excess  of  lead  removed  from  the  solution  by 
hydrogen  sulphide,  and  the  clear  liquid  thus  obtained  was 
submitted  to  dialysis.  The  above  bases  occurred  in  the 
dialysate.  In  the  non-dialyzable  portion  volatile  bases 
were  found  probably  identical  with  those  described  by 
Gautier  and  Etard. 

Tetanine,  C13H30N2O4,  was  obtained  in  1886  by 
Brieger  (III.,  94)  by  cultivating  impure  tetauus  microbes 
of  Rosenbach,  in  an  atmosphere  of  hydrogen  on  beef- 
broth  for  eight  days  at  37°-38°.  It  likewise  occurs  in 
cultures  on  brain-broth.  Later  (April,  1888),  Brieger 
succeeded  in  obtaining  tetanine  from  the  amputated  arm 
of  a  tetanus  patient,  identical  in  its  physiological  action 
and  chemical  reactions  with  that  isolated  from  cultures 
of  Rosenbach's  germs  on  beef-broth.  The  presence  of 
tetanine   during   life  in  tetanus   patients   has   thus   been 


266  BACTERIAL    POISONS. 

demonstrated.  It  has  not  been  found  in  the  brain  and 
nerve  tissue  of  persons  dead  from  tetanus.  A  portion  of 
the  jelly-like  mass  taken  from  the  amputated  arm  was 
found  to  contain  tetauus  bacilli  as  well  as  staphylococci 
and  streptococci,  and  when  planted  on  beef-broth,  tetanine 
was  formed,  but  no  tetanotoxine  or  spasmotoxiue. 

Kitasato  and  Weyl  (1890),  employing  pure  cultures 
of  the  tetanus  bacillus,  obtained  from  l|  kilogramme  beef 
used  as  culture  medium  1.7118  gramme  of  tetanine  hydro- 
chloride (0.137  per  cent.).     Tetanotoxine  was  also  present. 

For  its  isolation  Brieger  employed  the  following 
method :  The  cultures  were  slightly  acidulated  with 
hydrochloric  acid,  heated  and  filtered;  the  filtrate  was 
then  treated  with  lead  acetate  and  with  alcoholic  mercuric 
chloride  in  the  manner  described  under  mytilotoxine  (page 
255).  Kitasato  and  Weyl  digest  the  cultures  with  0.25 
per  cent,  hydrochloric  acid  for  some  hours  at  60°,  then  render 
slightly  alkaline,  filter,  and  distil  in  vacuo  at  60°.  The 
residue  in  the  retort  is  worked  for  tetanine  by  Brieger's 
method,  while  the  distillate  contains  tetanotoxine,  ammo- 
nia, indol,  hydrogen  sulphide,  phenol  and  butyric  acid. 
The  filtrate  from  the  above  mercuric  chloride  precipitate 
contains  the  greater  part  of  the  active  principle,  provided 
the  precipitate  has  been  thoroughly  washed.  After  the 
removal  of  the  mercury  by  hydrogen  sulphide,  it  is  evap- 
orated and  the  residue  is  repeatedly  extracted  with  absolute 
alcohol,  in  which  the  tetauus  poison  readily  dissolves,  and 
can  thus  be  separated  from  the  insoluble  ammonium 
chloride.  The  alcoholic  solution  is  treated  with  alcoholic 
platinum  chloride,  which  precipitates  the  ammonium  and 
creatinine  platinochlorides,  whilst  the  platinochloride  of  the 
poison  remains  in  solution.  The  filtrate  from  this  precipi- 
tate gives,  on  the  addition  of  ether,  a  flocculeut  precipitate 
possessing  exceedingly  deliquescent  properties.  The  pre- 
cipitate is,  therefore,  rapidly  filtered  off  by  means  of  a 
pump,  and  dried  in  vacuo.  It  can  then  be  recrystallized 
from  hot  96  per  cent,  alcohol,  and  the  beautiful  clear-yellow 
plates  thus  obtained,  if  dried  again  in  vacuo,  become 
rather  difficultly  soluble  in  water,  from  which  it  can  then 


CHEMISTRY    OF    THE    PTOMAINES.  267 

be  recrystallized  and  obtained  in  a  perfectly  pure  condi- 
tion. If  boiled  with  boneblack  it  decomposes,  yielding  a 
non-poisonous  crystalline  compound. 

Phosphomolybdic  acid  cannot  be  used  in  the  separation 
of  tetanine,  inasmuch  as  it  destroys  the  poison  (Brieger). 
Bocklisch  has  also  observed  that  it  destroys  the  poison 
formed  in  the  putrefaction  of  fish. 

Tetanine  obtained  by  treating  the  hydrochloride  with 
freshly  precipitated  moist  silver  oxide  forms  a  strongly 
alkaline  yellow  syrup.  With  alkaloidal  reagents  it  gives 
the  same  reactions  as  the  hydrochloride,  except  that  it  does 
not  give  a  blue  color  with  ferric  chloride  and  potassium 
ferricyanide.  It  is  easily  decomposed  in  acid  solution,  but 
is  permanent  in  alkaline  solution. 

The  Hydrochloride,  C]3H3UN204.2HC1,  is  deliques- 
cent and  is  easily  soluble  in  absolute  alcohol.  Beside  with 
platinum  it  combines  only  with  phosphomolybdic  acid  to 
form  an  easily  soluble  crystalline  precipitate,  which  on  the 
addition  of  ammonium  hydrate  becomes  white.  If,  how- 
ever, the  hydrochloride  is  impure,  phosphomolybdic  acid 
produces  a  precipitate  which  is  colored  an  intense  blue  by 
ammonia.  Potassium-bismuth  iodide  yields  a  precipitate 
which  is  at  first  amorphous,  but  soon  becomes  crystalline. 
Ferric  chloride  and  potassium  ferricyanide  produce  a  slowly 
developing  blue  color  which  probably  is  due  to  impurities. 

When  kept  for  some  months  the  highly  poisonous  hydro- 
chloride becomes  syrupy,  brownish,  and  wholly  inert. 
Examined  at  this  stage,  the  syrup  was  found,  by  means  of 
platinum  chloride,  to  contain  a  substance  the  hydrochlo- 
ride of  which  crystallized  in  plates.  This  is  readily  soluble 
in  water  and  alcohol,  and  melts  at  197°,  with  total  decom- 
position, the  same  as  tetanine.  It  combines  only  with  phos- 
phomolybdic acid  to  form  an  easily  soluble  compound.  The 
platinum  salt  has  the  composition  C6H13lSr02.2lICl.PtC]4. 
This  substance  is  non-poisonous  and  probably  an  amido- 
acid.  It  is  different,  however,  from  leucin  and  Nencki's 
isomers  of  leucin,  although  possessing  the  same  composi- 
tion. It  is  also  isomeric  with  mydatoxine,  C6H13N02,  but 
this  is  highly  poisonous  to  mice,  while  the  former  is  inert 


268  BACTERIAL    POISONS. 

(see   page    255).      Tetanine  resembles  nrytilotoxine  with 
respect  to  this  loss  of  toxicity  on  standing. 

The  Platinochloride,  C13H30N2O4.2HCl.PtCl4  (Pt  = 
28.33  per  cent.),  is  easily  soluble  in  absolute  alcohol  from 
which  it  is  precipitated  on  the  addition  of  ether.  From 
ninety-six  per  cent,  alcohol  it  crystallizes  in  clear  yellow 
plates.  After  repeated  recrystallization  from  alcohol  and 
drying  in  vacuo  it  becomes  difficultly  soluble  in  water  so 
that  it  can  be  recrystallized  from  the  latter.  It  decomposes 
at  197°. 

This  base  produces  the  characteristic,  though  probably 
not  all  the  symptoms  of  tetanus,  since  we  know  of  at  least 
three  other  toxines  (pages  194,  195)  which  occur  with  teta- 
nine  in  cultures  of  the  tetanus  microbe.  The  symptoms 
induced  by  relatively  large  doses  in  warm-blooded  animals, 
as  mice,  guinea-pigs,  and  rabbits,  exhibit  two  distinct  phases. 
In  the  first,  the  animal  is  thrown  into  a  lethargic  paralytic 
condition,  then  suddenly  becomes  uneasy,  and  the  respira- 
tion becomes  more  frequent.  This  is  followed  by  the  second 
phase,  in  which  tonic  and  clonic  convulsions,  especially  the 
former,  predominate  till  death  results.  0.5  gramme  has 
but  slight  action  on  guinea-pigs.  Small  doses  do  not 
seem  to  affect  guinea-pigs,  while  frogs  seem  to  be  much 
less  sensitive  than  mice.  The  characteristic  convulsions 
and  opisthotonus  seen  in  tetanus  in  man  are  also  produced 
in  guinea-pigs  on  injection  of  large  doses  of  this  base.  Dogs 
and  horses  seem  to  be  but  slightly  sensitive  to  the  action  of 
this  poison. 

A  Base,  C14N20lSr2O4,  was  isolated  by  Guareschi  in 
1887  from  putrid  fibrin.  It  occurs  in  the  chloroform  or 
ether  extracts  along  with  the  base  C10H13N,  and  is  probably 
an  amido-acid  (see  page  201). 

A  Base,  C7H18lSr206,  was  isolated  by  Pouchet  in  1884. 
It  is  said  to  form  short,  thick  prisms  which  become  brown 
when  exposed  to  light. 

The  Platijstochloride,  (C7H18N206.HCl)2PtCl4,  crys- 
tallizes in  prismatic  needles  which  are  insoluble  in  strong 


CHEMISTRY    OF    THE    PTOMAINES.  269 

alcohol.     For  further  details  in  regard   to  this  base  see 
page  265. 

Tyrotoxicon  has  been  obtained  in  poisonous  cheese 
(Vaughan,  Wallace,  Wolff),  in  poisonous  ice-cream 
(Vaughan,  JSTovy,  Schearer,  Ladd),  in  poisonous 
milk  (Vaughan,  Novy,  Newton,  Wallace,  Firth, 
Schearer),  and  in  cream-puffs  (Stanton).  The  methods 
of  separating  this  poison  and  its  effect  upon  animals  have 
already  been  given  with  sufficient  detail.  Chemically,  it  is 
very  instable.  When  warmed  with  water  to  about  90°,  it 
decomposes.  Hydrogen  sulphide  also  decomposes  it,  there- 
fore all  attempts  to  isolate  it  by  precipitation  with  some 
base,  such  as  mercury  or  lead,  and  then  removing  the  base 
with  hydrogen  sulphide,  have  failed.  Its  unstable  char- 
acter is  illustrated  by  the  fact  that  it  may  disappear 
altogether  within  twenty-four  hours  from  milk  rich  in  the 
poison  which  is  allowed  to  stand  in  an  open  beaker. 

With  potassium  hydrate  it  forms  a  compound  which 
agrees  in  crystalline  form,  chemical  reactions,  and  the  per 
cent,  of  potassium  which  it  contains,  with  the  compound  of 
diazobenzole  and  potassium  hydrate.  This  substance  is  best 
obtained  from  milk  containing  tyrotoxicon  as  follows  :  The 
filtered  milk,  which  is  acid  in  reaction,  is  neutralized  with 
sodium  carbonate,  agitated  with  an  equal  volume  of  ether, 
allowed  to  stand  in  a  stoppered  glass  cylinder  for  twenty- 
four  hours,  the  ether  removed,  and  allowed  to  evaporate 
spontaneously  from  an  open  dish.  The  aqueous  residue  is 
acidified  with  nitric  acid,  then  treated  with  an  equal  volume 
of  a  saturated  solution  of  potassium  hydrate,  and  the  whole 
concentrated  on  the  water-bath  (this  compound  is  not 
decomposed  below  130°).  On  beiug  heated  the  mixture 
becomes  yellowish-brown,  and  emits  a  peculiar  aromatic 
odor.  On  cooling  the  tyrotoxicon  compound  forms  in 
beautiful,  six-sided  plates  along  with  the  prisms  of  potas- 
sium nitrate. 

With  equal  parts  of  sulphuric  and  carbolic  acids,  pure 
tyrotoxicon  gives  a  green  coloration,  but  in  whey  the  color 
varies  from  yellow  to  orange-red.    This  color  reaction  may 


270  BACTEEIAL    POISONS. 

be  used  as  a  preliminary  test  in  examining  milk  for  tyro- 
toxicon. It  is  best  carried  out  as  follows  :  Place  on  a  clean 
porcelain  surface  two  or  three  drops  each  of  pure  carbolic 
and  sulphuric  acids.  Then  add  a  few  drops  of  the  aqueous 
solution  of  the  residue  left  after  the  spontaneous  evapora- 
tion of  the  ether.  If  tyrotoxicon  be  present,  a  yellow  to 
orange-red  coloration  will  be  produced.  This  test  is  to 
be  regarded  only  as  a  preliminary  one,  for  the  coloration 
may  be  due  to  the  presence  of  a  nitrate  or  nitrite,  or  as 
Huston  has  shown,  to  butyric  acid.  The  tyrotoxicon 
must  be  converted  into  the  potassium  compound  and  puri- 
fied before  the  absence  of  nitrate  or  nitrite  can  be  positively 
demonstrated.  Moreover,  the  physiological  test  should 
always  be  made  in  testing  for  this  poison. 

With  platinum  chloride  in  alcoholic  solution  tyrotoxicon 
forms  a  compound  which  explodes  with  great  violence  at 
the  temperature  of  the  water-bath.  This  also  corresponds 
with  the  compound  of  platinum  chloride  and  diazobenzole. 

Pure  tyrotoxicon  is  insoluble  in  ether,  and  its  extraction 
from  alkaline  solutions  by  this  solvent  is  due  to  the  pres- 
ence of  foreign  matter,  with  which  the  poison  is  taken  up 
by  the  ether. 

The  physiological  action  of  this  ptomaine  has  been  suf- 
ficiently discussed  in  a  preceding  chapter. 

Mydaleine  (/xvdaMog,  putrid)  is  a  poisonous  base  ob- 
tained in  1885  from  putrefying  cadaveric  organs,  liver, 
spleen,  etc.  (Briegee,  II.,  31,  48).  Though  it  is  appa- 
rently present  on  about  the  seventh  day,  it  is  unobtainable 
until  about  the  third  or  fourth  week.  The  method  for  its 
separation  from  the  accompanying  bases  is  given  under 
Sapriue  (page  220).  It  is  liable  to  occur  in  the  mercuric 
chloride  filtrate,  as  well  as  in  the  precipitate,  inasmuch  as 
the  double  salt  is  insoluble  only  in  perfectly  absolute  alco- 
hol. In  order  to  purify  the  platinochloride  obtained  as  on 
page  221,  it  is  repeatedly  recrystallized  from  a  very  small 
quantity  of  lukewarm  water.  This  base  has  not  been  ob- 
tained in  sufficient  quantity  to  permit  of  a  complete  deter- 
mination of  its  composition.     It  is  probably  a  diamine, 


CHEMISTRY    OF    THE    PTOMAINES.  271 

containing  four  or  five  carbon  atoms,  and  hence  is  nearly 
related  to  some  of  the  diamines  already  described. 

The  Platinochloride,  on  analysis,  gave  :  Pt  =  38.74 
C  =  10.83,  H  =  3.23.  It  crystallizes  in  small  needles,  and 
is  extremely  soluble  in  water. 

The  Hydrochloride  crystallizes  with  extreme  dif- 
ficulty, even  on  stauding  for  some  time  in  a  desiccator.  On 
exposure  to  the  air  it  rapidly  deliquesces. 

Physiological  Action. — Mydaleine  has  an  entirely  specific 
action.  Small  quantities  injected  into  guinea-pigs  or 
rabbits  produce,  after  a  short  time,  a  moistening  of  the 
under  lip,  and  an  abundant  flow  of  secretion  from  the  nose 
and  eyes.  The  pupils  dilate  gradually  to  maximum,  and 
become  reactionless ;  the  ear  vessels  become  strongly  in- 
jected, and  the  body  temperature  rises  1°  to  2°.  The  hairs 
bristle,  and  the  animal  occasionally  shudders.  Gradually 
the  salivation  ceases,  the  respiration  and  heart-action,  which 
were  at  first  hastened,  now  decrease,  the  temperature  falls, 
the  ears  become  pale,  and  the  animal  finally  recovers. 
During  the  action  of  the  poison  the  animal  shows  a  ten- 
dency to  sleep,  and  the  peristaltic  action  of  the  intestines  is 
heightened.  Larger  doses  (0.050  gramme)  induce  an  ex- 
ceedingly violent  action,  which  invariably  results  in  the 
death  of  the  animal.  On  post-mortem,  the  heart  is  found 
to  be  stopped  in  diastole,  and  the  intestines  and  bladder 
contracted  ;  otherwise  nothing  abnormal  is  observed. 

A  Toxic  Base. — From  human  livers  and  spleens  which 
were  decomposing  for  two  weeks  in  thorough  contact  with 
air  there  was  isolated,  besides  cadaverine  and  putrescine,  a 
small  quantity  of  a  poisonous  base  (Briegee,  II.,  29,  48). 
The  mercuric  chloride  precipitate  was  decomposed,  and  the 
hydrochlorides  were  precipitated  by  gold  chloride  (to  re- 
move cadaverine,  which  is  soluble),  and  the  aurochloride 
was  then  changed  into  the  platinum  salt,  whereby  the  in- 
soluble putrescine  platinochloride  was  removed.  In  the 
mother-liquors  from  the  putrescine  salt  an  easily  soluble 
platinum  compound  was  separated,  and  found  to  contain 
41.30  per  cent.  Pt.     It  crystallized  in  fine  needles.     The 


272  BACTERIAL    POISONS. 

hydrochloride  formed  small,  readily  deliquescent  needles, 
and  did  not  produce  a  precipitate  in  alcoholic  platinum 
chloride.  Injected  into  guinea-pigs  and  rabbits  it  induced 
au  exalted  peristaltic  action  of  the  intestiues,  which  lasted 
several  days,  and  produced  in  the  animals,  on  account  of 
the  continuous  evacuations,  a  condition  of  great  weakness. 
No  disturbance  in  the  functions  of  the  other  organs  was 
observed. 

A  Base  was  isolated  from  decomposing  haddock  which 
were  exposed  for  five  days  during  summer  in  an  open  iron 
vessel.  Briegee  (L,  42)  found  in  the  aqueous  mercuric 
chloride  precipitate  (see  page  258)  a  base  the  hydrochloride 
of  which  crystallized  in  well-formed,  small  needles.  The 
platinochloride  likewise  crystallized  in  beautiful  needles, 
and  gave,  on  analysis,  36.03  per  cent,  of  Pt;  7.81  per  cent, 
of  N. 

A  substance  of  muscarine-like  action  was  obtained  by 
Brieger  (I.,  59)  from  putrefying  gelatin,  ten  days  at 
35°,  though  in  insufficient  quantity  to  permit  a  determina- 
tion of  its  character.  The  residue  containing  this  substance 
gave,  on  distillation  with  alkali,  only  ammonia. 

A  Base  was  obtained  by  Bocklisch  (III.,  52,  53)  from 
herring  which  had  undergone  putrefaction  fur  twelve  days. 
It  was  found  in  the  distillate,  together  with  trimethylamine 
and  dimethylamine,  obtained  by  distilling  the  mercuric 
chloride  filtrate,  after  the  removal  of  the  mercury,  with 
sodium  hydrate.  The  platiuochloride  was  easily  soluble, 
and  crystallized  in  large  thin  plates.  On  analysis  it  gave  : 
Pt  =  28.57,  C  =  22.34,  H  =  4.66.  The  hydrochloride  is 
easily  soluble  in  water,  and  in  absolute  alcohol,  and  be- 
sides with  platinum  gives  only  with  phosphomolybdic  acid 
a  yellow  precipitate  which  is  soluble  in  excess,  and  with 
ammonia  gives  an  immediate  blue  color.  It  immediately 
reduces  a  mixture  of  ferric  chloride  and  potassium  ferri- 
cyanide   with    formation    of  Berlin    blue ;   and   similarly 


CHEMISTEY    OF    THE    PTOMAINES.  273 

throws    down     metallic    gold    from    solutions    of    gold 
chloride. 

From  poisonous  mussel,  Brieger  (III.,  79)  obtained  an 
aurochloride  of  a  base  crystallizing  in  needles.  The  quan- 
tity isolated  was  insufficient  for  analysis.  It  is  interesting 
because  of  its  property  of  inducing  salivation,  a  symptom 
which  has  been  observed  by  Schmidtmann  and  by  Crumpe 
in  some  cases  of  mussel  poisoning. 

A  Base  was  obtained  by  Guareschi  and  Mosso  (Joum. 
far  praktische  Ohem.,  28,  508)  from  fresh  beef,  in  the 
alkaline  ether  extract  obtained  by  Dragendorff's  method. 
It  formed  a  yellowish  alkaline  fluid,  of  uupleasant  odor, 
and  after  a  time  gave  a  deposit  of  microscopic  crystals. 
The  hydrochloride  gave  the  following  reactions  :  Gold  chlo- 
ride, yellow  crystalline  precipitate  ;  platinum  chloride,  pre- 
cipitate ;  potassium  iodide  and  iodine  in  hydriodic  acid, 
kermes-red  precipitate ;  phosphotungstic  acid,  nothing ; 
phosphomolybdic  acid,  au  abundant  yellow  precipitate;  tan- 
nic acid,  heavy,  grayish  precipitate,  same  with  Mayer's 
reagent  ;  picric  acid,  yellow  precipitate;  MarmiVs  reagent, 
precipitate  soluble  in  excess  ;  potassium  bichromate,  noth- 
ing ;  potassium  permanganate  and  sulphuric  acid,  violet 
color  ;  potassium  ferricyanide  and  ferric  chloride,  Prussian 
blue  precipitate. 

By  giving  a  precipitate  with  tannin,  and  not  with  phos- 
photungstic acid,  it  resembles  neurine. 

Ch.  Gram  has  studied  the  decomposition  of  yeast  under 
the  influence  of  an  infusion  of  hay.  The  yeast  was  allowed 
to  putrefy  for  fourteen  days,  and  was  then  treated  with  zinc 
sulphate.  The  latter  was  precipitated  by  barium  hydrate, 
and  the  filtrate  after  the  removal  of  the  barium  by  sul- 
phuric acid,  was  evaporated  to  dryness,  and  extracted  with 
absolute  alcohol.  The  alcoholic  solution  was  evaporated, 
and  the  residue  again  extracted  with  alcohol.  The  extrac- 
tion residue  was  taken  up  with  water,  and  again  subjected  to 
the  above  treatment  with  zinc  sulphate,  barium  hydrate,  etc. 


274  BACTERIAL    POISONS. 

The  filtrate  was  poisonous,  and  produced,  in  frogs,  paral- 
ysis and  stoppage  of  the  heart  in  diastole  Addition  of 
platinum  chloride  and  alcohol  precipitated  two  bases.  One 
of  these,  although  possessing  a  curara-like  action,  did  not 
aifect  the  heart.  When  its  solution  was  heated  for  twenty- 
four  hours  on  the  water-bath,  it  caused  general  paralysis 
and  stoppage  of  the  heart.  The  platinochlori.de  contained 
38.05  per  cent,  of  platinum. 

The  other  base  also  possessed  a  slight  curara-like  action, 
and  its  platinochloride  gave,  on  analysis,  40.92  and  39.4 
per  cent,  of  platinum. 

Beieger  found  a  basic  substance  in  small  quantities  in 
cultures  of  the  staphylococcus  pyogenes  aureus  on  bouillon 
and  beef-broth  (II.,  74).  The  hydrochloride  formed  groups 
of  colorless,  non-deliquescent  needles.  With  platinum 
chloride  it  yielded  a  double  salt,  crystallizing  in  needles, 
and  containing  32.93  per  cent,  of  Pt.  For  its  reactions, 
see  Table  I. 

From  aqueous  as  well  as  alcoholic  solutions  of  cultures  of 
staphylococcus  aureus  Leber  (1888)  isolated  a  crystalline 
substance  which  he  named  phlogosine.  The  composition 
of  this  substance  is  not  known.  It  does  not  seem  to  con- 
tain nitrogen,  and  inasmuch  as  it  blackens  silver  it  prob- 
ably contains  sulphur.  It  crystallizes  in  fine  needles  which 
are  soluble  in  ether  and  in  alcohol ;  difficultly  soluble  in 
water.  It  sublimes  in  needles.  Alkalies  precipitate  it  as 
amorphous  yellow  floccules  which  are  soluble  in  acid  and 
then  can  be  recrystallized.  With  potassium  ferricyanide 
and  ferric  chloride  it  yields  a  blue  color,  and  with  potassio- 
mercuric,  cadmic,  and  bismuth  iodides  precipitates  which 
are  soluble  in  excess.  No  precipitate  is  produced  by  gold 
or  platinum  chlorides,  phosphotungstic  or  molybdic,  tan- 
nic or  picric  acids. 

A  small  quantity  applied  to  the  conjunctiva  produces 
intense  inflammation,  suppuration,  and  necrosis.  Intro- 
duced into  the  anterior  chamber  it  induces  intense  suppura- 
tion and  keratitis.  ■  The  substance  is  entirely  distinct  from 
the  base  obtained  by  Brieger,  described  above. 


CHEMISTRY    OF    THE    PTOMAINES.  275 

A  Base — boiling  point  about  284° — was  obtained  by 
Brieger  (II.,  61)  from  human  livers  and  spleens  which 
were  putrefying  for  two  to  three  weeks.  It  occurs  in  the 
mercuric  chloride  filtrate,  as  described  under  Saprine,  page 
220,  together  with  some  mydaleine,  trimethylamine,  and 
hydrocarbons.  The  filtrate,  after  the  mercury  is  removed 
by  hydrogen  sulphide,  is  evaporated  to  dryness,  and  finally 
the  last  traces  of  water  are  removed  in  a  vacuum.  The 
residue  is  then  treated  with  absolute  alcohol,  and  from  this 
alcoholic  solution  the  mydaleine  is  precipitated  by  the  addi- 
tion of  alcoholic  mercuric  chloride.  The  trimethylamine 
is  separated  by  distillation  of  the  alkaline  filtrate,  previously 
deprived  of  its  mercury  by  hydrogen  sulphide ;  while  the 
mother-liquor  yields  an  oily  mixture  of  hydrocarbons  and 
bases.  The  latter  were  separated  by  fractional  distillation, 
whereby  only  one  of  the  bases  was  obtained  iu  sufficient 
quantity  for  study.  It  boiled  at  about  284°,  and  gave 
with  hydrochloric  acid,  on  evaporation,  a  salt  crystallizing 
in  beautiful,  long  needles,  which  were  very  easily  soluble 
in  perfectly  absolute  alcohol.  With  gold  chloride  and  picric 
acid  it  gave  only  oily  products ;  with  ferric  chloride  and 
potassium  ferricyanide,  an  intense  blue;  with  platinum 
chloride,  an  extremely  easily  soluble  double  salt,  which 
appeared  under  the  microscope  in  the  form  of  very  fine 
needles,  while  from  alcohol- ether  the  double  salt  slowly 
separated  in  thin  plates  which  contained  30.36  per  cent,  of 
platinum.  The  free  base  showed  a  slight  fluorescence.  It 
is  not  poisonous,  and,  according  to  Brieger,  is  probably 
a  pyridine  derivative. 

Other  non-poisonous  bases  were  present  in  very  small 
quantity  in  the  mother-liquor  described  above,  after  the 
separation  of  the  oily  mixture. 

Peptotoxine. — By  this  name  Brieger  (I.,  14-19)  has- 
designated  a  poisonous  base  which  he  has  found  in  some 
peptones,  and  hence  in  the  digestion  of  fibrin  ;  in  putre- 
fying albuminous  substances,  such  as  fibrin,  casein,  brain, 
liver,  and  muscles.  It  is  a  well-known  fact  that  animal 
tissues,  in  the  early  stages  of  putrefaction,  possess  strong 
toxic  properties,  even  before  the  decomposition  could  have 


276  BACTERIAL    POISONS. 

advanced  far  enough  to  efi'eet  a  splitting-up  of  the  proteid 
and  carbohydrate  molecules.  Brieger  and  others  have 
tried  to  seek  an  explanation  of  this  toxicity  by  connecting 
it  with  an  early  peptonization  of  the  proteids  brought  about 
by  the  action  of  ferments  which  are  distributed  throughout 
the  tissues,  and  which  begin  their  activity  immediately  after 
death.  This  poison  has  not  been  definitely  isolated,  but  its 
general  properties  and  action  have  been  studied  by 
Brieger  and  Salkowski.  The  former  prepared  it  by 
digesting  fibrin  for  twenty-four  hours  with  gastric  juice  at 
the  temperature  of  the  blood.  The  perfectly  fresh  peptone 
thus  obtained  was  evaporated  to  a  syrupy  residue,  and  this 
was  then  extracted  with  boiling  alcohol.  The  residue  left 
on  evaporation  of  the  alcoholic  solution  was  digested  for 
some  time  with  amyl  alcohol,  which  on  subsequent  evapor- 
ation gave  amorphous  brownish  masses.  This  extract  can 
then  be  purified  by  neutral  lead  acetate.  The  filtrate,  after 
the  removal  of  the  lead  by  hydrogen  sulphide,  is  repeat- 
edly extracted  with  ether,  then  evaporated  to  dryness,  and 
extracted  as  before,  with  amyl  alcohol.  This  final  extract 
is  evaporated  to  drive  off  the  alcohol,  taken  up  with  water, 
and  filtered.  The  colorless  aqueous  solution  thus  obtained 
contains  the  poisonous  substance,  which,  however,  can  only 
with  extreme  difficulty  be  brought  to  crystallization  in 
vacuo. 

This  poison,  when  in  its  purest  condition,  as  shown  by 
its  failure  to  give  the  biuret  reaction,  possesses  a  neutral 
reaction.  Its  behavior  to  Millon's  reagent  is  quite  charac- 
teristic: it  gives  a  white  precipitate,  which  on  boiling 
becomes  intensely  red.  From  this  reaction,  Brieger  is 
inclined  to  regard  this  substance  as  a  hydroxyl  or  an 
amido-derivative  of  benzole.  The  ptomaine  can  be  ex- 
tracted from  acid  as  well  as  alkaline  solution  by  amyl 
alcohol — more  difficult  in  the  cold  than  on  heating.  It 
is  absolutely  insoluble  in  ether,  benzol,  and  chloroform  ; 
very  soluble  in  water.  It  is  not  destroyed  by  boiling,  by 
passing  hydrogen  sulphide,  or  by  strong  alkalies;  but  is 
destroyed,  however,  when  the  putrefaction  lasts  longer 
than  eight  days.  For  its  behavior  to  reagents,  see 
Table  I. 


CHEMISTRY    OF    THE    PTOMAINES.  277 

Various  observers  have  shown  that  peptone  possesses  a 
toxic  action,  and  some  have  been  led  to  regard  this  toxicity 
as  not  due  to  the  peptone  itself,  but  rather  to  the  presence 
of  this  or  some  other  ptomaine.  At  least  Brieger  found 
one  specimen  of  dry  Witte's  peptone  to  be  perfectly  harm- 
less ;  whereas,  the  fresh  peptone  formed  by  fibrin  digestion 
possessed  strong  toxic  powers.  Moreover,  this  non- 
poisonous  peptone  when  exposed  to  the  action  of  gastric 
juice  was  found  to  yield  the  poisonous  substance.  The 
poisonous  nature  of  proteids  and  the  physiological  action 
of  this  base  will  be  described  later. 

Pyocyanine,  C14H14N02,  is  the  coloring  matter  of  blue 
pus,  and  is  produced  by  the  action  of  bacillus  pyocyaneus. 
It  was  isolated  by  Ledderhose  (1887)  and  is  said  to  be  an 
anthracene  derivative.  On  contact  with  the  air  it  is  oxidized 
to  pyoxanthose,  a  yellow  substance.  According  to  Kunz 
it  contains  nitrogen  and  sulphur.  The  picrate  is  of  a  dark 
reddish-brown  color ;  the  platinum  salt  is  black  and  some- 
times is  obtained  as  glittering  fine  golden  needles. 


13 


278 


BACTERIAL    POISONS. 


Table  of  Ptomaines. 


Formula. 

Name. 

Discoverer. 

Physiological  action.1 

C   H5N 

Methylamine. 

Bocklisch. 

Nou-poisonous. 

C2H7N 

Dimethylamine. 

Brieger. 

"            " 

C3H9N 

.Trimethylamine. 

Dessaignes. 

"            " 

C2H6N 

Spermine(?). 

Kunz. 

"            " 

C2H7N 

Bthylamine. 

Hesse. 

"            " 

C4  HUN 

Diethylamine. 

Bocklisch. 

"            *' 

C6  H16N 

Triethylamine. 

Brieger. 

"            " 

C3H9N 

Propylamine. 

" 

C4  HnN 

Butylamine. 

Gautier  &  Mourgues. 

Poisonous(?). 

C6  HnN  (?) 

Tetanotoxine. 

Brieger. 

Poisonous. 

C6  H13N 

Amylamine. 

Hesse. 

" 

C6  H16N 

Hexylamlne. 

" 

" 

C7  HUN 

Di-hydrolutidine. 

Gautier  &  Mourgues. 

" 

C8  HnN 

Collidine(V). 

Nencki. 

C8  HUN 

Pyridine  base(?). 

0.  de  Coninck. 

C8  H13N 

Hydrocollidine(?). 

Gautier  and  Etard. 

Poisonous. 

C9  H13N 

ParVoline(?). 

"        "         " 

010H15N 

Unnamed. 

Guareschi  and  Mosso. 

Poisonous. 

CioH16N 

Pyridine  base(?). 

0.  de  Coninck. 

Ci0H17N 

Hydrocoridine(?). 

Griffiths. 

C32H31N 

Unnamed. 

Delezinier. 

C2  H3  N2 

Ethylidenediamine(?). 

Brieger. 

Poisonous. 

C3  H8  N2 

Trimethylenediamine(?). 

" 

" 

C4  H12N2 

Putrescine. 

" 

Not  very  poisonous. 

C6  H14N2 

Cadaverine. 

" 

"               " 

C5  H14N» 

Neuridine 

" 

Non-poisonous. 

C6  H14N2 

Saprine. 

" 

"            " 

C7  H10N2 

Unnamed. 

Morin. 

"            " 

C10H26N2(?) 

Susotoxine. 

Novy. 

Poisonous. 

C2  H7  N3 

Methyl-guanidine. 

Brieger. 

" 

C19H27N3 

Morrhuine. 

Gautier  &  Mourgues. 

Diuretic,  etc. 

Ci3H20N4 

Unnamed. 

Oser. 

Ci7Ha8N4 

" 

Gautier  and  Etard. 

C.25H32N4 

Aselline. 

Gautier  &  Mourgues. 

Poisonous. 

C5  H13N  0 

Neurine. 

Brieger. 

" 

08  HnN  0 

My  dine. 

" 

Non-poisonous. 

C6  HnN  02 

b-amido-valerianic  acid. 

E.  and  H.  Salkowski. 

tc 

C5  H15N  02 

Choline. 

Brieger. 

Poisonous. 

C6  H13N  02 

Mydatoxine. 

' 

C6H13N02 

Unnamed. 

Brieger,  1888. 
(tetanus  cult.) 

Non-poisonous. 

C6  H15N  02 

Mytilotoxine. 

Brieger. 

Poisonous. 

1  Only  those  bases  are  here  denoted  as  poisonous  which  possess  a  decided  toxicity. 


CHEMISTRY    OF    THE    PTOMAINES, 


279 


Table  op  Ptomaines — Continued. 


Formula. 

Name. 

Discoverer. 

Physiological  action.1 

C7  H17N  02 

Gadinine. 

Brieger. 

Poisonous. 

C7  H17N  02 

Typhotoxine. 

" 

" 

C7  H17N  02 

Unnamed. 

" 

" 

C14H14N  02 

Pyocyanine. 

Ledderhose. 

Non-poisonous. 

C5  H13N  03 

Betaine. 

Brieger. 

" 

C5  H15*N  03 

Muscarine. 

" 

Poisonous. 

C9  HlgN  03 

Morrhuic  acid. 

Gautier  &  Mourgues. 

C5  H12N204 

Unnamed. 

Pouchet. 

Poisonous. 

Ci3H3oN204 

Tetanine. 

Brieger. 

" 

O14H20N2O4 

Unnamed. 

Guareschi. 

C7  H18N206 

" 

Pouchet. 

Poisonous. 

Tyrotoxicon. 

Vaughan. 

Mydaleine. 

Brieger. 

Spasmotoxine. 

" 

A  diamine(?). 

"    (tetanus  cult.) 

Peptotoxine. 

" 

Phlogosine. 

Leber. 

Inflammatory. 

1  Only  those  bases  are  here  denoted  as  poisonous  which  possess  a  decided  toxicity. 


CHAPTER  XII. 

CHEMISTEY   OF   THE   LEUCOMAINES. 

Under  this  head  are  classed  those  basic  substances  which 
are  found  in  the  living  tissues,  either  as  the  products  of 
fermentative  changes  or  of  retrograde  metamorphosis. 
Most  of  these  substances  have  already  been  known  for 
many  years,  though  their  real  significance  as  alkaloidal 
bodies,  and  their  relation  to  the  functional  activities  of  the 
animal  organism  have  been  but  little  understood,  or  rather 
they  have  not  been  brought  together  under  the  leading 
conception  that  they  are  alkaloidal  products  of  physiologi- 
cal change.  The  first  attempt  at  the  systematic  study  and 
generalization  of  these  basic  substances  was  made  by 
Gautier,  who  applied  to  them  the  name  leucomaines,  a 
term  derived  from  the  Greek  Aed^w^a,  signifying  white  of 
eggs.  Under  this  name  he  includes  all  those  basic  sub- 
stances which  are  formed  in  animal  tissues  during  normal 
life,  in  contradistinction  to  the  ptomaines  or  basic  products 
of  putrefaction.  The  distinction  between  vegetable  and 
animal  alkaloids  is  not  very  well  defined,  and,  in  fact,  there 
seem  to  be  reasons  for  considering  their  formation  as  due 
to  the  same  causes  which  bear  an  intimate  relation  to  the 
physiology  of  the  cells  and  tissues  of  both  kingdoms. 
Thus,  vegetable  tissues  are  known  to  contain  not  only 
definite  ptomaines,  such  as  choline,  but  also  leucomaines,  as 
hypoxanthine,  xanthine,  etc.  Indeed,  in  this  latter  group 
must  be  placed,  on  account  of  their  relation  to  xanthine, 
those  well-defined  alkaloidal  bases,  caffeine  and  theobro- 
mine. Not  only  are  the  representatives  of  these  two 
divisions  of  basic  substances  common  to  both  kingdoms, 
but  their  parent  bodies,  lecithin,  nuclein,  etc.,  are  known 
to  occur  in  both,  thus  giving  rise  to  the  same  bases  on 
decomposition. 


CHEMISTRY    OF    THE    LEUCOM  AINES .  281 

So  far  as  the  genesis  of  most  of  the  leucoma'ines  is 
concerned,  we  know  very  little,  though  Gautier  is  of  the 
belief  that  they  are  being  formed  continuously  and  inces- 
santly in  the  animal  tissues  side  by  side  with  the  forma- 
tion of  urea  and  carbonic  acid  and  at  the  expense  of  the 
nitrogenous  elements.  It  is  quite  probable,  as  Kossel  has 
pointed  out,  that  some  of  these  products  are  in  themselves 
antecedents  of  end-products  of  metabolism.  This  is  unques- 
tionably true  of  the  imido  group,  which  exists  in  the  ade- 
nine and  guanine  molecules,  and  through  vital  or  putre- 
factive processes  is  split  off,  giving  rise  to  ammonia,  which 
in  turn  serves  to  form  urea  and  uric  acid.  Bouchard  has 
sought  an  explanation  of  the  presence  of  these  bases  in  the 
urine,  by  supposing  that  they  were  originally  formed  in 
the  intestinal  tract,  from  which  they  were  absorbed  into  the 
system,  to  be  subsequently  eliminated  by  the  kidneys.  This 
view  has  also  been  brought  forward  by  Schar  (1886),  who 
holds  that  these  bases,  which  may  be  formed  by  putrefactive 
changes  in  the  intestinal  tract,  are  absorbed  into  the  circu- 
latory system,  whence  they  may  be  partly  eliminated  by 
the  kidneys  or  may  be  partly  deposited  in  the  tissues  them- 
selves. 

The  views  of  Bouchard  and  Schar  have,  to  a  certain 
extent,  been  confirmed  by  the  investigations  of  Udranszky 
and  Baumann,  who  showed  that  the  well-known  ptomaines, 
cadaverine  and  putrescine,  occur  in  the  urine  in  cystinuria, 
and  are  formed  by  putrefactive  changes  induced  in  the  in- 
testinal tract  probably  by  specific  micro5rganisms.  Under 
this  same  head  fall  the  recent  observations  of  Wolkow 
and  Baumann,  that  alkapton  is  produced  from  ty  rosin  by 
similar  changes  in  the  intestines.  The  origin  of  the  true 
leucomames  cannot,  however,  be  accounted  for  in  this 
manner,  for  they  are  indissolubly  connected  with  the  meta- 
bolism of  the  cell  itself,  and  are,  therefore,  formed  in  the 
tissues  and  organs  proper,  especially  those  rich  in  nucleated 
cells. 

Another  source  of  the  nitrogenous  bases  must  not  be 
lost  sight  of,  and  that  is  protoplasm  itself.  The  researches 
of  Drechsel,  Siegfried,  and  Schulze  have  shown  that 


282  BACTERIAL    POISONS. 

nitrogenous  bases  do  result  from  the  decomposition  of 
animal  and  vegetable  proteids  (see  p.  242). 

The  leucomaines  proper  can  be  divided  into  two  distinct 
and  well-defined  groups :  (1)  the  Uric  Acid  Group,  and 
(2)  the  Creatinine  Group. 

The  first  of  these  divisions  contains  a  number  of  well- 
known  bases  which  are  closely  related  to  uric  acid.  The 
order  in  which  they  will  be  described  is  as  follows  : 

Adenine,  CgHgNg. 

Hypoxanthine,  C5H4lSr40. 

Guanine,  C5H5N50. 

Xanthine,  05H4N4O2. 

(Uric  Acid,  C5H4N403.) 

Heteroxanthine,  C6H6lSr402. 

Paraxanthine,  C7H8N402. 

Carnine,  C7H8N403. 

Pseudoxanthine,  C4H5N40. 

Gerontine,  C5H14N2. 

Spermine,  C2H5N  (?). 

The  members  of  the  second  group  have  all  been  dis- 
covered by  Gautier,  and  by  him  are  regarded  as  allied 
to  creatine  and  creatinine.  These  two  substances,  especially 
the  latter,  have  been  hitherto  regarded  as  strongly  basic  in 
character,  but  Salkowski  has  recently  shown  that  creati- 
nine, when  perfectly  pure,  possesses  little  or  no  alkaline 
reaction,  and,  moreover,  does  not  combine  with  acids.  The 
bases  in  this  group  are  : 

(Creatinine,  C4H7N30.) 

(Creatine,  C4H9N302.) 

Cruso- creatinine,  C5H8N40. 
Xantho-creatinine,  C5H10N4O. 

Amphi-creatine,  C9H1cN704. 

Base,  CnH;4N10O5. 

Base,  C12H25Nn05. 

Besides  these  two  general  classes  of  leucoma'ines,  there 
may  be  made  a  third  class  of  undetermined  leucoma'ines, 


GHEMISTKY    OF    THE     LEUCOM  AINES .  283 

embracing  those  bases  which  have  been  observed,  but 
studied  more  or  less  incompletely,  in  the  various  physio- 
logical secretions  of  the  body. 

LeUCOMAINES    OF    THE    UeIC    A.CID    GROUP. 

Adenine,  C5H5N~5,  which  was  discovered  by  Kossel  in 
1885,  forms  the  simplest  member  of  the  uric  acid  group  of 
leucomames,  and  as  such  it  deserves  special  attention,  inas- 
much as  it  shows  most  clearly  the  relation  that  exists 
between  hydrocyanic  acid  and  the  members  of  this  group. 
This  base  is  apparently  formed  by  the  polymerization  of 
hydrocyanic  acid — a  view  that  is  confirmed,  at  least  in 
part,  by  the  fact  that  on  heating  with  potassium  hydrate 
to  200°,  it  yields  a  large  quantity  of  potassium  cyanide. 
Moreover,  by  the  action  of  reducing  agents,  it  is  converted 
into  a  substance  similar  to,  if  not  identical  with,  azulmic 
acid.  It  has  not  been  prepared  synthetically,  though 
Gautier  has  claimed  to  have  synthesized  two  closely  re- 
lated bodies,  xanthine  and  methyl-xanthine,  by  simple 
heating  of  hydrocyanic  acid  in  a  sealed  tube  in  contact  with 
water  and  a  little  acetic  acid. 

This  base  was  first  prepared  from  pancreatic  glands — 
hence  the  term  adenine,  which  is  derived  from  the  Greek 
word  adyv,  meaning  a  gland.  It  has  since  been  shown  to 
occur  together  with  guanine,  hypoxanthine,  etc.,  as  a 
decomposition-product  of  nuclein,  and,  therefore,  it  may 
be  obtained  from  all  tissues  and  organs,  animal  or  vege- 
table, rich  in  nucleated  cells.  Accordingly,  it  has  been 
found  in  the  kidneys,  spleen,  pancreatic,  thymus  and  lym- 
phatic glands,  in  beer-yeast,  in  spermatic  fluids,  but  not  in 
testicles  of  the  steer  ;  occurs  also  in  tea-leaves.  In  the  latter 
adenine  appears  to  exist  in  a  preformed  condition,  since  it  can 
be  extracted  without  the  use  of  acid  reagents.  The  thymus 
gland,  as  a  prototype  of  embryonic,  highly  cellular  tissue, 
yields  a  considerable  amount  of  adenine  ;  that  from  a  calf, 
for  instance,  was  found  by  Schindler  to  contain  0.18  per 
cent.     It  has  also  been  observed  in  the  liver  and  urine  of 


284  BACTERIAL    POISONS. 

leucocytheemic  patients  ;  its  occurrence  in  this  disease  will 
be  readily  understood  when  it  is  remembered  that  leucocy- 
thsemia  is  characterized  by  the  presence  in  the  blood  of  an 
unusual  proportion  of  the  nucleated  white  blood-corpuscles, 
which,  owing  to  various  unfavorable  conditions,  become  de- 
stroyed in  time,  and  the  contained  nuclein,  as  a  result, 
splits  up  into  adenine  and  guanine.  These  two  bases  may, 
therefore,  be  expected  in  all  pathological  conditions  where 
there  is  an  abnormal  accumulation  of  pus.  Indeed,  as  early 
as  1865,  Naunyn  extracted  from  pus,  obtained  from  the 
pleural  cavity,  a  considerable  quantity  of  a  substance 
which  was  probably  either  adenine  or  guanine,  or  both. 
Adenine  does  not  occur,  or  only  in  minute  traces,  in  meat 
extract ;  and  in  this  it  resembles  guanine,  which  is  present 
only  in  traces.  This  may  be  due  to  the  fact  that  adenine 
and  guanine  are  readily  converted  into  hypoxanthine  and 
xanthine  respectively,  as  has  been  shown  in  the  putrefaction 
experiments  of  Schindler.  They  may  be  considered  as 
transitional  products  of  cell-metabolism,  the  imido  group 
contained  in  each  readily  being  replaced  by  oxygen,  and 
giving  rise  to  ammonia,  and  this  in  turn  to  urea.  Kossel, 
however,  explains  this  fact  on  the  ground  that  the  muscle 
tissue  is  very  poor  in  nucleated  cells,  i.  e.,  in  nuclein.  It 
would  seem  that  the  muscle  cell  in  losing  the  morphological 
character  of  a  cell  has  also  suifered  a  corresponding  loss  in 
its  chemical  properties.  For  while  the  decomposition- 
products  of  nuclein — hypoxanthine,  xanthine,  phosphoric 
acid,  etc. — are  found  in  the  muscle  tissue,  they  do  not  exist 
in  combination  as  they  do  in  the  nuclein  molecules.  This  is 
seen  in  the  fact  that  the  bases  exist  in  the  free  condition, 
since  they  can  be  extracted  by  water ;  and  again,  the  phos- 
phoric acid  is  present  in  the  muscle  tissue,  not  in  organic 
combination,  but  as  a  salt.  In  the  nucleated  cell,  adenine, 
guanine,  etc.,  do  not  exist  in  the  free  condition,  but  form, 
in  part  at  least,  with  albumin  and  phosphoric  acid,  a  loose 
combination  which  is  readily  decomposed  by  the  action  of 
acids  at  the  boiling  temperature.  This  same  change  takes 
place  spontaneously  after  death. 

There  can  be  no  doubt  that  adenine  and  guanine  play  an 


CHEMISTRY    OF    THE     LEUCOMAINES.         285 

important  part  in  the  physiological  function  of  the  cell 
nucleus,  which,  from  recent  observations,  appears  to  be 
necessary  to  the  formation  and  building  up  of  organic 
matter.  It  is  now  known  that  non-nucleated  cells,  though 
capable  of  living,  are  not  capable  of  reproduction.  We 
must  look,  therefore,  to  the  nucleus  as  the  seat  of  the 
functional  activity  of  the  cell — indeed,  of  the  entire  organ- 
ism. Nuclein,  the  parent  substance  of  adenine  and  guanine, 
is  the  best  known  and  probably  most  important  constituent 
of  the  nucleus,  and  as  such  it  has  been  already  credited 
with  a  direct  relation  to  the  reproductive  powers  of  the 
cell.  This  chemical  view  has  recently  been  confirmed  by 
Zacharias,  who  showed  that  chromatin  of  histologists  is 
identical  with  nuclein.  Liebejrmann  has  questioned  nu- 
clein as  being  the  source  of  xanthine  compounds,  but  in 
this  he  is  not  supported  by  the  mass  of  evidence. 

The  method  employed  by  Kossel  for  the  preparation 
of  adenine,  is  as  follows :  The  finely  divided  pancreatic 
glands  are  heated  to  boiling,  for  three  or  four  hours,  with  a, 
large  quantity  of  dilute  sulphuric  acid  (0.5  per  cent,  by 
volume  of  concentrated  acid),  and  the  acid  solution  thus 
obtained  is  treated  with  a  slight  excess  of  hot  concentrated 
baryta  water.  The  excess  of  baryta  is  removed  by  carbonic 
acid,  and  the  solution  is  then  filtered ;  the  filtrate  is  con- 
centrated to  a  small  bulk,  about  100  c.c,  rendered  alkaline 
with  ammonium  hydrate,  and  finally  precipitated  with  an 
ammoniacal  solution  of  silver  nitrate.  The  precipitate, 
consisting  of  the  silver  compound  of  the  xanthine  bodies, 
is  partially  dried  by  spreading  over  porous  porcelain 
plates ;  then  dissolved  in  warm  nitric  acid  of  specific 
gravity  1.1,  to  which  a  little  urea  has  been  added  to  pre- 
vent the  formation  of  hypoxanthine  should  traces  of  nitrous 
acid  be  present.  The  filtered  acid  solution,  treated  with 
silver  nitrate,  on  cooling,  gives  a  deposit  of  the  silver 
salts  of  hypoxanthine,  guanine,  and  adenine,  which  is  fil- 
tered oft  and  thoroughly  washed.  The  adenine  separates 
out  almost  quantitatively  if  a  little  silver  nitrate  solution 
is  added.  The  filtrate  contains  any  xanthine  silver  com- 
pound that  may  be  present.     The  washed  precipitate  of  the 

13* 


286  BACTERIAL    POISONS. 

silver  salts  is  suspended  in  water,  nitric  acid  added,  decom- 
posed with  hydrogen  sulphide  (ammonium  sulphide,  or, 
better,  hydrochloric  acid,  may  be  used),  and  the  clear  filtrate 
is  concentrated  on  the  water-bath  to  a  small  volume.  It  is 
then  saturated  with  ammonium  hydrate  and  digested  on  the 
water-bath  for  some  time,  whereby  adenine  and  hypoxan- 
thine  go  into  solution,  while  the  guanine  remains  undis- 
solved (see  p.  287).  From  the  ammoniacal  solution  on 
partial  concentration  and  subsequent  cooling,  the  adenine 
crystallizes  out  first,  whereas  the  more  soluble  hypoxanthine 
remains  in  solution.  If  the  adenine  is  still  colored  it  can 
be  purified  by  dissolving  in  water  and  boiling  with  animal 
charcoal.  The  hot  aqueous  solution  is  then  rendered  very 
slightly  alkaline  with  ammonium  hydrate  and  allowed  to 
cool ;  adenine  crystallizes  out,  and  can  be  still  further  puri- 
fied by  recrystallization  from  water. 

Ammonium  sulphide  has  been  employed  by  Schindlee, 
in  place  of  hydrogen  sulphide,  in  decomposing  the  silver 
compounds  of  the  above  bases.  Beuhns  recommends  in- 
stead warming  with  very  dilute  hydrochloric  acid,  espe- 
cially if  guanine  is  present.  The  solution  can  then  be 
neutralized  with  JSTaHC03,  using  methyl-orange  as  indi- 
cator, and  the  adenine  separated  from  hypoxanthine  by  the 
picric  acid  method  described  below. 

Another  method  for  the  separation  of  adenine  from 
hypoxanthine  is  based  upon  the  behavior  of  the  nitrates 
of  these  bases  in  aqueous  solution.  From  concentrated 
aqueous  solutions  of  the  nitrates,  free  hypoxanthine  crystal- 
lizes out  first,  because  the  nitrate  is  decomposed;  whereas, 
adenine,  which  is  a  stronger  base,  remains  in  combination 
with  the  acid,  in  solution. 

Schindeer  determines  adenine  and  hypoxanthine  indi- 
rectly. The  ammoniacal  solution  which  is  filtered  from  the 
insoluble  guanine  is  evaporated  to  dryness  on  a  weighed 
platinum  dish,  dried  at  110°,  and  weighed.  A  nitrogen 
determination  is  now  made  of  the  mixed  bases  and  from 
these  data  the  proportion  of  each  is  calculated. 

By  far  the  best  method  for  the  quantitative  separation  of 
adenine  and  hypoxanthine  is  the  picrate  method  of  Bruhns. 


CHEMISTRY    OF    THE    LEUCOM AINES .  287 

The  solution  of  the  salts  of  the  bases,  preferably  as  nitrates 
or  sulphates,  must  be  neutral  or  faintly  acid  ;  excess  of 
alkali  or  acid  interferes.  Such  a  solution  can  be  obtained 
by  evaporating  the  filtrate  from  the  guanine  in  Kossel's 
method  (page  286),  and  dissolving  the  residue  in  nitric 
acid ;  this  is  neutralized  with  sodium  carbonate,  using 
methyl-orange  as  indicator.  On  the  addition  of  excess  of 
sodium  picrate  the  adenine  is  thrown  down  as  a  clear  yel- 
low flocculent  precipitate.  If  the  precipitation  is  made  at 
the  boiling  temperature,  on  cooling  the  adenine  salt  sepa- 
rates in  a  crystalline  condition  and  is  more  easily  filtered 
and  washed.  After  standing  fifteen  minutes  the  precipitate 
is  filtered  off  by  the  aid  of  a  suction-pump  on  a  weighed 
filter,  washed  with  cold  water,  and  dried  at  100°.  As  a 
correction  for  the  solubility  of  the  adenine  picrate,  2.4  mg. 
per  100  c.c.  filtrate  can  be  added  to  the  calculated  amount 
of  adenine. 

The  hypoxanthine  picrate  is  very  soluble,  and,  therefore, 
remains  in  solution.  In  this  it  is  estimated  according  to 
the  method  described  on  page  302. 

Adenine,  when  crystallized  from  warm  or  impure  solu- 
tions, is  obtained  either  as  an  amorphous  substance,  pearly 
plates,  or  in  the  form  of  very  small  microscopic  needles  ; 
from  dilute  cold  solutions  it  separates  in  long,  needle-shaped 
crystals  containing  three  molecules  of  water.  This  water 
of  crystallization  is  lost  on  exposure  to  the  air  or  on  heating 
to  53°,  and  the  crystals  become  opaque.  It  is  soluble  in 
about  1086  parts  of  water  at  the  ordinary  temperature ; 
more  easily  in  hot  water,  from  which,  on  cooling,  it  recrys- 
tallizes.  The  aqueous  solution  possesses  a  neutral  reaction. 
The  free  base  is  insoluble  in  ether,  chloroform,  and  alcohol ; 
soluble  in  glacial  acetic  acid,  and  somewhat  in  hot  alcohol. 
It  dissolves- readily  in  mineral  acids,  yielding  well-crystal- 
lizable  salts.  The  fixed  alkalies  dissolve  it  with  ease,  but 
on  neutralization  of  the  solution  it  is  reprecipitated  In 
aqueous  ammonium  hydrate  it  is  more  readily  soluble  than 
guanine  (which  is  insoluble,  Schindlee),  and  more  diffi- 
cultly soluble  tha>n  hypoxanthine — a  fact  which  is  made  use 


288  BACTERIAL    POISONS. 

of  to  effect  a  separation  from  these  bases.     It  is  but  slightly 
soluble  in  sodium  carbonate. 

Adenine  can  be  heated  to  278°  without  melting ;  at  this 
temperature  it  becomes  slightly  yellow,  and  yields  a  white 
sublimate.  It  can  be  completely  volatilized  without  decom- 
position, by  heating  on  an  oil-bath  to  220°  ;  the  sublimate 
consists  of  pure,  white,  plumose  needles  of  adenine,  but  at 
250°  partial  decomposition  occurs,  and  some  hydrocyanic 
acid  forms.  When  heated  with  potassium  hydrate  to  200° 
on  an  oil-bath,  it  yields  a  considerable  quantity  of  potas- 
sium cyanide.  Adenine  is  quite  indifferent  to  the  action  of 
acids,  alkalies,  and  even  oxidizing  agents.  Thus,  it  may  be 
boiled  for  hours  with  baryta,  potash,  or  hydrochloric  acid, 
without  suffering  decomposition.  But  when  heated  with 
dilute  hydrochloric  acid,  or  concentrated  hydriodic  acid, 
in  a  sealed  tube  at  a  temperature  exceeding  100°,  adenine 
is  completely  decomposed,  with  formation  of  carbonic  acid 
and  ammonia  : 

C5H5N5  +  5H20  +  50  =  5C02  +  5KH3. 

The  free  base,  as  well  as  benzoyl-adenine,  is  unaffected  by 
the  weak  oxidizing  action  of  potassium  permanganate,  but 
on  stronger  oxidation  it  is  wholly  destroyed.  Bromine  water 
produces  in  aqueous  solutions  of  adenine  an  oily  precipitate, 
which,  on  contact  with  potassium  hydrate  or  ammonia,  gives 
a  beautiful  red  or  violet  color.  Sodium  amalgam  and  zinc 
chloride  appear  to  have  no  action;  but  on  boiling  with  zinc 
and  hydrochloric  acid  it  yields  a  very  unstable  reduction- 
product,  which  in  the  presence  of  oxygen  first  assumes  a  red 
color,  and  finally  throws  down  a  reddish-brown  precipitate. 
This  brown  substance  appears  to  be  identical  with  azulmic 
acid,  which  has  been  known  for  a  long  time  as  a  product  of 
the  polymerization  of  hydrocyanic  acid. 

When  adenine  is  evaporated  on  the  water-bath  with  dilute 
or  fuming  nitric  acid,  it  gives  a  white  residue  which  fails  to 
give  any  coloration  with  sodium,  ammonium,  or  barium 
hydrate.  Similarly,  it  does  not  give  the  so-called  Weidel's 
reaction  (murexide  test)  on  evaporation  with  chlorine  water 
and  exposure  of  the  residue  to  an  ammoniacal  atmosphere. 


CHEMISTRY    OF    THE     LEUCOMAINES.  '289 

In  this  respect  it  resembles  hypoxanthine,  which,  when 
pure,  does  not  answer  to  either  of  these  tests.  Another  test 
for  adenine,  which,  however,  is  given  also  by  hypoxanthine 
but  not  by  guanine  and  caffeine,  is  as  follows :  The  sub- 
stance to  be  tested  is  digested  for  half  an  hour  with  zinc 
and  hydrochloric  acid  in  a  test-tube  on  the  water-bath.  If 
adenine  is  present,  the  solution  will  assume  on  standing, 
more  rapidly  on  shaking,  a  ruby-red  coloration,  which  later 
on  turns  into  a  brownish-red.  This  reaction  depends  upon 
the  formation  of  a  reduction-product,  which,  owing  to  its 
unstable  nature,  is  soon  oxidized  by  the  oxygen  of  the 
atmosphere  into  a  brownish,  amorphous  substance,  appa- 
rently identical  with  azulmic  acid. 

On  treatment  with  nitrous  acid,  it  is  converted  into  hypo- 
xanthine according  to  the  equation  : 

C5H5N5  +  HN02  =  05H4N4O  +  N2  +  H20. 

This  formation  of  hypoxanthine  from  adenine  is  analogous 
to  Strecker's  transformation  of  guanine  into  xanthine  by 
a  similar  action  of  nitrous  acid  (see  Guanine).  In  both 
cases  the  change  of  a  highly  nitrogenized  into  a  less  nitro- 
genized  body  is  accomplished  by  replacing  an  NH  group 
by  O,  or,  more  exactly,  of  an  NH2  group  by  OH.  In  fact, 
the  change  is  identical  with  that  seen  in  the  conversion 
of  primary  amines  into  primary  alcohols.     Thus, 

C2H5.NH2  +  HN02  =  C2H5OH .+  N2  +H20. 

Ethylamine.  Ethyl  Alcohol. 

In  the  extraction  of  adenine  from  the  mother-liquors  of 
tea-leaves  after  removal  of  caffeine,  if  urea  is  not  added  to 
the  nitric  acid,  nearly  one-half  of  the  adenine  may  be  con- 
verted into  hypoxanthine.  By  processes  of  putrefaction 
adenine  is  converted  into  hypoxanthine  and  guanine  into 
xanthine  (Schindler).  The  change  is,  therefore,  some- 
what analogous  to  that  produced  by  nitrous  acid.  Adenine 
undergoes  this  decomposition  much  more  rapidly  than  the 
other  xanthine  compounds. 

The  ease  with  which  adenine  and  guanine  are  reduced 
outside  of  the  organism  shows  that  similar  changes  may  take 


290  BACTERIAL    POISONS. 

place  within  the  cell-nucleus  proper.  For  we  know  that 
every  cell  is  endowed  with  an  enormous  reducing  power, 
and  hence  it  is  not  difficult  to  see  how  the  oxygen-free 
adenine  can  be  readily  converted  into  a  body  or  bodies 
which  greedily  take  up  oxygen.  We  must,  therefore,  look 
upon  adenine  and  guanine  not  only  as  the  antecedents  of 
hypoxanthine  and  xanthine,  but  also  as  intermediate  pro- 
ducts which,  when  they  form  in  the  cell,  may  give  rise  to 
important  chemical  processes,  especially  those  of  a  synthetic 
nature.  It  is  highly  probable  that  the  study  of  the  decom- 
position-products of  nuclein  will  explain  to  us  many  of  the 
metabolic  changes  in  the  organism,  and  throw  additional 
light  upon  the  migration  of  the  amido  group  from  the 
proteid  molecule  to  the  amido  acids  and  urea  derivatives. 
Thus,  the  formation  of  xanthine  from  guanine  represents 
the  conversion  of  a  guanidine  residue  into  a  urea  residue. 
A  similar  change  is  undoubtedly  effected  in  the  transforma- 
tion of  adenine  into  hypoxanthine. 

Adenine  unites  with  bases,  acids,  and  salts.  The  salts 
of  adenine  with  mineral  acids  can  be  recrystallized,  thus 
differing  from  the  corresponding  salts  of  guanine  and  hypo- 
xanthine, which  are  dissociated  by  the  action  of  water. 
The  solutions  of  the  salts,  however,  show  an  acid  reaction 
to  litmus  but  not  to  methyl-orange. 

The  hydrochloride,  C5H5N5.HC1  +  |H20,  forms  color- 
less, transparent,  strongly  refracting  crystals.  One  part  of 
the  anhydrous  salt  is  soluble  in  41.9  parts  of  cold  water. 
M  icroscopically  it  is  distinct  from  that  of  hypoxanthine  and 
adenine-hypoxanthine. 

The  nitrate,  C5H5N5.HN03  +  JH20,  crystallizes  from 
the  aqueous  solution  in  fine,  stellate  needles.  One  part  of 
the  dry  salt  dissolves  in  110.6  parts  of  water. 

The  sulphate,  (C5H5N5)2.H2S04  +  2H20,  can  be  obtained 
from  the  aqueous  solution  in  two  different  crystalline  forms. 
This  may  possibly  be  due  to  the  presence  of  adenine-hypo- 
xanthine compound  (Beuhns).  It  is  easily  soluble  in  hot 
water,  and  at  the  ordinary  temperature  it  is  soluble  in  1 53 
parts  of  water. 

The  oxalate,  C5H5]Sr6.C2H204  +  H20,  is  obtained  by  dis- 


CHEMISTRY    OF    THE     LEUCOM AINES.         291 

solving  adenine  in  hot,  dilute,  aqueous  oxalic  acid,  from 
which  solution,  on  cooling,  it  separates  as  a  voluminous, 
difficultly  soluble  precipitate  of  roundish  masses  which  are 
composed  of  long,  delicate  needles.  The  oxalates  of  guanine, 
hypoxanthine,  and  xanthine  are  more  easily  soluble  than 
that  of  adenine,  and  exhibit,  moreover,  a  different  appear- 
ance. 

The  picrate,  C5H5N5.C6H2(N02)3OH  +  H20,  is  thrown 
down  as  a  bright  yellow  flocculent  precipitate,  when  aqueous 
solutions  of  adenine  salts  are  treated  with  sodium  picrate. 
Recrystallized  from  hot  water  it  forms  bright-yellow,  very 
voluminous  bunches  of  long  fine  needles,  which,  on  drying, 
acquire  a  silky  lustre  and  form  a  felted  mass.  It  is  diffi- 
cultly soluble  in  cold  water  (1  :  3500) ;  more  readily  in  hot 
water  and  in  alcohol  (96  per  cent.) ;  is  insoluble  in  dilute 
acids.  The  water  of  crystallization  is  not  lost  on  exposure 
to  air  but  is  driven  off  at  100°  ;  the  salt  then  remains  un- 
changed even  at  220°.  A  cold  concentrated  aqueous  solu- 
tion of  the  salt  treated  with  one-tenth  its  volume  of  cold  con- 
centrated solution  of  sodium  picrate  produces  a  precipitate 
of  short  fine  needles  consisting  of  most  of  the  adenine  picrate 
(five-sevenths).  The  solubility  of  the  picrate  can  thus  be 
reduced  to  as  low  as  1  :  13750,  and  on  this  fact  is  based  the 
quantitative  method  of  Bruhns.  The  salt  can  also  be 
obtained  in  its  characteristic  groups  by  combining  cold 
saturated  aqueous  adenine  solution  (1  :  1086)  with  picric 
acid ;  with  sodium  picrate,  however,  adenine  gives  no  pre- 
cipitate, since  the  picrate  is  soluble  in  an  equivalent  quan- 
tity of  sodium  hydrate.  Thus  is  explained  Kossel's 
statement  that  adenine  forms  an  easily  soluble  compound 
with  picric  acid.  Heated  on  a  platinum  foil  it  burns  slowly 
and  leaves  considerable  carbon  residue.  The  very  bright 
yellow  color  of  the  salt  serves  to  distinguish  it  from  most 
of  the  other  picrates,  especially  guanine  picrate. 

The  platinochloride,  (C5H5N5.HCl)2PtCl4,  crystallizes 
from  dilute  aqueous  solution  in  small  yellow  needles.  The 
concentrated  aqueous  solution  of  this  salt,  when  boiled  for 
some  time,  decomposes,  with   the   separation  of  a  clear, 


292  BACTERIAL    POISONS. 

yellow  powder,  which  is  but  slightly  soluble  in  cold  water, 
and  has  the  composition  C5H5N5.HCl.PtCl4. 

The  aurochloride,  on  evaporation,  yields  very  charac- 
teristic forms. 

The  silver  salt  of  adenine,  C5H4AgN6,  is  formed  when 
silver  nitrate  is  added  in  molecular  proportion  to  a  boiling 
ammoniacal  solution  of  adenine.  An  excess  of  silver 
nitrate  produces,  in  the  cold,  the  compound  C5H3Ag2N5  + 
H20,  which  is  converted  slowly  in  the  cold,  immediately  on 
warming,  into  the  other  salt,  according  to  the  equation  : 

2(C5H3Ag2N5  +  H20)  =  2C5H4AgN5  +  Ag20  +  H20. 

Owing  to  this  instability  the  two  compounds  are  always 
found  together  in  varying  proportion.  Both  are  difficultly 
soluble  in  water,  and  ammonia  even  at  the  boiling-point. 
The  precipitation  of  adenine  by  an  ammonical  silver  solu- 
tion is  complete,  and  is  therefore  available  for  quantitative 
estimation. 

Adenine  silver  nitrate,  C5H5N5.  AgN03,  (Ag  =  35.4  per 
cent.),  corresponds  to  the  similar  hypoxanthine  and  guanine 
salts.  It  is  obtained  by  dissolving  the  above  silver  com- 
pounds in  hot  nitric  acid ;  and  from  this  solution,  on  cool- 
ing, it  separates  in  needle-shaped  crystals,  which  are  not 
permanent.  This  lack  of  stability,  as  compared  with  the 
permanent  hypoxanthine  silver  nitrate,  was  first  pointed 
out  by  Kossel,  and  was  thought  to  be  due  to  loss  of  nitric 
acid  in  washing,  and  also  by  heating  at  100°.  Bruhns, 
however,  has  shown  that  the  acidity  of  the  wash-water  is 
indicated  by  litmus,  but  not  by  methyl-orange,  which  is 
not  colored  red  by  silver  nitrate.  The  reaction  is,  there- 
fore, due  not  to  free  nitric  acid,  but  to  silver  nitrate.  It 
would  seem  that  adenine,  as  well  as  hypoxanthine,  and  pos- 
sibly xanthine,  form  silver  compounds  containing  one  and 
two  molecules  of  silver  nitrate ;  the  greater  the  quantity  of 
silver  nitrate  used  the  higher  is  the  per  cent,  of  silver,  i.e., 
the  more  of  the  latter  compound  is  formed.  These  are  very 
unstable,  and  are  decomposed  by  dilute  nitric  acid,  more  so 
by  water,  into  silver  nitrate  and  the  compound  containing 
one  molecule  of  silver  nitrate.     We  have  in  this  behavior 


CHEMISTRY    OF    THE    LEUCOMAINES.  293 

an  interesting  case  of  mass-action  and  chemical  equilibrium 
between  adenine,  silver  nitrate,  nitric  acid  and  water. 
Ammonium  hydrate  removes  the  nitric  acid  from  this  as 
easily  as  from  the  hypoxanthine  compound,  and  there  is 
formed,  according  to  the  composition  of  the  original  salt,  a 
varying  mixture  of  C5H4AgN~5  and  C5H3Ag2N5  +  H20. 
The  solubility  in  nitric  acid  is  about  the  same  as  that  of 
hypoxanthine  silver  nitrate. 

Adenine  silver  picrate,  C5H4AgN5.C6H2(NO2)30H  + 
H20,  is  obtained  as  an  amorphous  voluminous  yellow  pre- 
cipitate when  silver  nitrate  is  added  to  a  cold  aqueous  solu- 
tion of  adenine  picrate.  If  the  latter  solution  is  previously 
raised  to  the  boiling-point  the  precipitate  then  soon  becomes 
crystalline  and  rapidly  subsides.  The  adenine  can  thus  be 
almost  wholly  removed  from  solution.  The  crystalline 
form  loses  its  water  of  crystallization  at  120°,  while  the 
amorphous  form  does  not  appreciably  decrease  in  weight  and 
its  composition  does  not  appear  to  be  as  constant  as  that  of 
the  corresponding  hypoxanthine  compound.  On  treatment 
with  ammonium  hydrate  the  picric  acid  is  removed,  and 
adenine  silver,  C5H4AgN5,  is  left,  stained  yellow  by  traces 
of  picric  acid. 

Aclenine-mercury  picrate,  (C5H4N5)2Hg.2C6H2(N02)3OH, 
can  be  prepared  by  treating  a  hot  concentrated  aqueous 
solution  of  adenine  picrate  with  an  excess  of  sodium  picrate 
and  then  with  mercuric  chloride.  It  forms  a  yellow  gran- 
ular crystalline  precipitate  (microscopic  needles)  which  rap- 
idly subsides  and  increases  in  quantity  as  the  solution  cools. 
Its  composition  apparently  varies,  containing  one  to  two 
molecules  of  water,  according  to  the  temperature  of  the 
solution.  One  molecule  is  given  off  at  100°,  and  the  second 
at  105°-120°.  The  latter  preparation,  then,  on  exposure  to 
the  air,  rapidly  absorbs  one  molecule  of  water.  The  ob- 
ject of  the  sodium  picrate  in  the  precipitation  is  to  combine 
with  the  hydrochloric  acid,  which  is  set  free.  The  precipi- 
tate produced  by  mercuric  chloride  in  cold  adenine  picrate 
solution  shows  yellow  and  white  granules,  and  is  not  homo- 
geneous. Bruhns  considers  it  to  be  a  mixture  of  the  aden- 
ine-mercury  picrate  and  the  compound  C5H4N5Hg2Cl3 ;  if 


294  BACTERIAL    POISONS. 

sodium  picrate  is  added,  however,  the  pure  adenine-mercury 
picrate  forms,  since  no  hydrochloric  acid  is  set  free. 

Adenine-mercuric  chloride,  C5H4N5HgCl,  is  thrown  down 
as  a  white,  finely  granular  precipitate  when  a  boiling  aque- 
ous adenine  solution  is  treated  gradually  with  concentrated 
mercuric  chloride  solution.  It  is  formed  according  to  the 
following  reaction  : 

C5H5N5  +  HgCl2  =  C5H  AHgCl  +  HC1. 

That  free  hydrochloric  acid  forms  can  be  ascertained  by 
methyl  orange.  Treated  with  ammonium  hydrate  the 
chlorine  is  removed,  and  there  is  formed  apparently  the 
compound  C5H4N5HgOH.  If  dissolved  iu  warm  dilute 
hydrochloric  acid  and  allowed  to  crystallize,  the  double  salt 
C5H5N5.HCl.HgCl2  +  2H20  separates  in  long  stellate  silky 
needles. 

Another  mercury  compound,  C5H4N5Hg2Cl3,  is  obtained 
when  the  precipitation  takes  place  in  the  cold.  The 
precipitate  is  white,  flocculent,  and  anhydrous.  In  this 
reaction,  as  above,  for  each  adenine  molecule  an  equivalent 
of  hydrochloric  acid  is  set  free.  This  same  body  is  also 
produced  when  an  adenine  solution  is  boiled  with  a  large 
excess  of  mercuric  chloride  and  as  little  hydrochloric  as 
possible  to  effect  solution.  On  cooling  small  stellate  needles 
separate  out,  which  do  not  lose  their  weight  at  110°.  It 
can  also  be  obtained  by  boiling  the  following  compounds 
with  water. 

When  adenine  is  boiled  with  a  large  excess  of  mercuric 
chloride  and  much  hydrochloric  acid  to  completely  dissolve 
the  precipitate  that  first  forms,  there  is  deposited  on  cooling 
a  crystalline  product,  which  is  variable  in  its  composition, 
and  apparently  consists  of  double  salts  of  adenine  and 
mercuric  chloride,  such  as  C5H5N5.HC1.5HgCl2  and  C5H5N"5. 
HC1.6HgCl2.  On  boiling  with  water  these  rapidly  de- 
compose, forming  the  compound  C5H4N5.Hg2Cl3.  The 
formation  of  a  double  salt,  C5H5N5.HCl.HgCl2  +  2H20 
is  described  above. 

Adenine-mercury  cyanide,  (C5H5N5)2Hg(CN)2,  separates 


CHEMISTRY    OF    THE    LEUCOM AINES .  295 

as  stellate  needles  and  plates  when  ■  a  mixture  of  hot  solu- 
tions of  adenine  and  mercuric  cyanide  are  allowed  to  cool. 

An  adenine  bismuth  iodide,  C5H5N5.HI.2BiI3  +  2H20, 
is  obtained  when  an  aqueous  adenine  solution  is  treated 
with  potassium  bismuth  iodide  containing  free  hydriodic 
acid.  The  heavy  precipitate,  which  in  color  resembles 
carbon  monoxide  haemoglobin,  consists  of  microscopic  glit- 
tering red  needles.  On  contact  with  much  water  it  partly 
decomposes,  forming  light  reddish-yellow  amorphous  floc- 
cules,  which  become  darkish-brown  at  100°. 

Adenine  bromide.  By  treating  well-dried  adenine  with 
excess  of  dried  bromine  a  dark-red  body  is  obtained  which 
appears  to  contain  six  atoms  of  bromine.  On  mere  ex- 
posure to  the  air,  more  rapidly  on  heating  at  100°-120°, 
it  decomposes,  yielding  bromine  and  a  brom-adenine, 
C5H4BrN5.  This  compound  is  white,  difficultly  soluble  in 
cold  water  (1  :  10,000),  more  readily  in  hot  water,  very 
easily  in  ammonia.  It  crystallizes  from  water  or  dilute 
ammonia  in  stellate  needles.  It  is  a  rather  strong  base  and 
forms  well-characterized  salts  from  which  it  is  thrown 
down  as  a  white  micro-crystalline  precipitate  by  addition 
of  ammonia.  It  is  also  formed  from  adenine- bromide  by 
treatment  with  sodium  bisulphite.  The  picrate  resembles 
that  of  adenine  but  is  more  voluminous ;  silver  compounds 
are  also  formed  resembling  those  of  adenine.  The  silver 
nitrate  compound  decomposes  on  boiling  with  nitric  acid 
with  separat:on  of  silver  bromide.  It  is  only  difficultly 
attacked  by  boiling  alcoholic  potash. 

When  adenine  is  treated  with  zinc  and  hydrochloric  acid, 
in  the  cold,  it  forms  a  difficultly  soluble  crystalline  double 
salt  which  has  not  been  obtained  in  the  pure  state.  This 
double  salt  is  not  obtained  by  direct  treatment  of  adenine 
hydrochloride  with  zinc  chloride. 

One  of  the  hydrogen  atoms  of  adenine  is  capable  of  re- 
placement by  organic  radicals.  Thus  it  forms  crystalline 
methyl  and  ethyl  compounds. 

Acetyl-adenine,  C5H4N5.CO.CH3,  can  be  obtained  by 
heating  the  anhydrous  base  with  an  excess  of  acetic  anhy- 
dride for  some  time,  in  an  oil-bath,  at  130°.     It  crystallizes 


296  BACTERIAL    POISON'S. 

in  small  white  scales  which  dissolve  but  slightly  in  cold 
water  and  in  alcohol ;  more  readily  in  hot  water,  in  dilute 
acids  and  alkalies.  Heated  to  260°  it  becomes  yellow  but 
does  not  melt. 

Benzoyl-adenine,  C5H4N5.CO.C6H„  is  obtained  by  the 
action  of  benzoic  anhydride,  but  not  of  benzoyl  chloride, 
on  adenine.  It  crystallizes  from  water  in  long,  lustrous, 
thin  needles  which  sometimes  are  grouped  in  bundles,  and 
melt  at  234°-235°.  It  is  easily  soluble  in  hot  alcohol,  from 
which  it  recrystallizes  on  cooling ;  also  in  dilute  acids  and 
in  ammonia.  With  ammoniacal  silver  nitrate  it  gives  a 
precipitate  resembling  that  of  adenine,  but  is  more  readily 
soluble  in  ammonia.  This  compound  is  quite  stable,  since 
it  decomposes  very  slowly  on  boiling  with  hydrochloric 
acid ;  on  protracted  boiling  with  water  it  is  changed  into 
adenine  and  benzoic  acid. 

Benzyl-adenine,  C5H4N5.CH2.C6H5,  was  obtained  by 
Thoiss  by  heating  well-dried  adenine  with  benzyl  chloride 
to  boiling  (178°)  on  an  oil-bath.  The  compound  forms 
pure  white  microscopic  crystals  and  melts  at  259°.  It  is 
easily  soluble  in  hot  water  and  in  hot  alcohol.  With  acids 
it  forms  salts  from  which  alkalies  throw  down  the  base. 
The  hydrochloride  forms  fine  glossy  needles  which  are 
readily  soluble  in  alcohol  and  in  water,  but  not  in  ether. 
The  sulphate  and  nitrate  possess  similar  properties.  Like 
adenine  it  yields  a  silver  compound  which  is  insoluble  in 
ammonia.  On  reduction  with  zinc  and  hydrochloric  acid 
it  forms  an  amorphous  red  unstable  compound.  Treated 
with  nitrous  acid,  benzyl-adenine  is  reduced  to  benzyl-hypo- 
xanthine,  thus  showing  that  the  benzyl  group  replaces  a 
hydrogen  atom  in  the  group  C5H4N4,  which  Kossel  has 
called  adenyl  (see  page  307). 

Benzyl-adenine  picrate,  C12HnlN"5.C6H2(N02)3OH,  is  ob- 
tained as  fine  felted  yellow  needles,  which  are  fairly  soluble 
in  water  and  in  alcohol ;  insoluble  in  ether. 

A  methyl-adenine  was  obtained  by  Thoiss  in  an  impure 
state  by  heating  dried  adenine  with  methyl  iodide  in  a  sealed 
tube  at  100°.  It  can  be  crystallized  from  absolute  alcohol. 
The  aqueous  solution  of  the  base  is  precipitated  by  baryta 


CHEMISTRY    OF    THE     LEUCO  M  AINES.  297 

water ;  alcoholic  zinc  chloride  also  yields  a  precipitate  which 
is  soluble  in  excess  of  ammonium  hydrate.  Mercuric  nitrate 
also  gives  a  precipitate.  Cadmium  chloride  yields  a  pre- 
cipitate which  dissolves  on  warming,  reappears  on  cooling, 
and  is  soluble  in  ammonia.  Basic  lead  acetate  has  no  effect. 
Nothing  definite  is  known  in  regard  to  the  physiological 
action  of  adenine,  except  that  when  fed  to  dogs  it  appears 
to  be  eliminated  as  such,  in  part  at  least,  by  the  urine. 

Adenine-Hypoxanthine,  C5H5N5  -f-  C5H4N40.  The 
occurrence  of  this  compound  was  observed  by  Kossel,  but 
it  was  isolated  and  studied  for  the  first  time  by  Bruhns. 
It  can  be  prepared  by  cooling  a  hot  aqueous  solution  of 
equal  parts  of  the  two  bases.  At  first  it  is  obtained  as 
thick,  starch-like  semi-transparent  masses,  which  later  in 
part  become  white  and  chalky.  By  spontaneous  evapora- 
tion of  its  solution  in  very  dilute  ammonia  it  forms  pearly 
aggregates  of  very  small  radially  arranged  needles,  which 
contain  water  of  crystallization.  These  effloresce  some- 
what and  lose  the  water  at  100°.  The  compound  is  more 
readily  soluble  in  water  than  its  components,  but  an  exact 
determination  of  its  solubility  is  impossible,  inasmuch  as 
the  separation  from  hot  solutions  is  not  completed  for  some 
weeks.  Any  adenine  present  can  be  separated  by  recrys- 
tallization.  It  forms  a  distinct  crystalline  hydrochloride, 
which  should  be  borne  in  mind  when  examining  microscopic- 
ally for  the  two  bases ;  but  the  combination  is  loose,  since 
addition  of  gold  chloride  brings  down  the  characteristic 
gold  salt  of  adenine.  Ordinarily  it  does  not  form  salts 
with  sulphuric  or  nitric  acids,  but  more  often  is  decomposed 
by  these,  so  that  the  difficultly  soluble  adenine  crystallizes 
out.  Once,  however,  Bruhns  obtained  a  sulphate  which 
differed  from  the  pure  adenine  and  hypoxanthine  sulphates  ; 
thus  is  perhaps  explained  the  observation  of  Kossel  that 
adenine  sulphate  forms  crystals  belonging  to  two  systems. 
The  compound  can  be  decomposed  into  its  constituents  by 
fractional  crystallization  of  the  sulphate  or  nitrate ;  but 
better  by  forming  the  picrates,  which  are  very  unequally 
soluble  in  water.    The  existence  of  this  compound  undoubt- 


298  BACTERIAL    POISONS. 

edly  explains  many  of  the  mistakes  and  discrepancies  con- 
cerning the  properties  of  hypoxanthine,  which  it  resembles 
more  than  adenine,  and  for  the  same  reason,  perhaps, 
adenine  was  so  often  overlooked. 

Hypoxanthine,  05H4lSr4O,  sometimes  also  known  as 
sarcine  or  sarkine,  was  discovered  by  Scherer  (1850)  in 
splenic  pulp  and  in  the  muscles  of  the  heart,  and  was 
named  thus  because  it  contains  one  atom  of  oxygen  less 
than  xanthine.  It  has  since  been  obtained,  usually  accom- 
panying adenine  and  guanine,  from  nearly  all  of  the 
animal  tissues  and  organs  rich  in  nucleated  cells,  i.  e.,  in 
nuclein.  It  has  been  found  in  blood  after  death,  but  not 
in  blood  when  flowing  through  the  bloodvessels.  Salomon 
has  recently  shown  it  to  be  a  normal  constituent  of  urine, 
present,  however,  in  an  exceedingly  minute  quantity.  In 
the  blood  and  urine  of  leucocythsemic  patients  it  occurs  in 
increased  quantity  owing  to  the  abnormally  large  number 
of  nucleated  white  blood-corpuscles  in  circulation  (see  page 
284).  Bence  Jokes  observed  in  the  urine  of  a  boy,  who 
about  three  years  before  showed  the  symptoms  of  renal 
colic,  a  deposit  of  characteristic  whetstone-like  crystals, 
resembling  uric  acid,  but  differing  from  the  latter  by  dis- 
solving readily  on  the  application  of  heat,  while  from 
hydrochloric  acid  it  crystallized  in  elongated  six-sided 
plates.  These  crystals  he  believed  to  be  those  of  xan- 
thine, but  Scherer  and  others  consider  them  to  be  hypo- 
xanthine. It  is  therefore  quite  possible,  though  very  rare, 
for  this  base  to  form  a  deposit  in  the  urine  and  to  be 
confounded  in  shape  with  uric  acid.  Thudichum  has 
obtained  it  from  the  urine  of  persons  sick  with  liver  or 
kidney  diseases. 

Among  other  places  it  has  been  found  in  the  brain, 
muscle,  serum,  marrow  of  bones,  kidney,  heart,  spleen, 
liver,  peripheral  muscles  (sarkine  of  Strecker)  ;  in  the 
spawn  of  salmon  (Piccard),  in  the  testicles  of  the  bull 
(Salomon),  in  the  nuclein  of  pus  and  red  corpuscles  (Kos- 
SEL),  in  developing  eggs,  and  in  putrefaction  of  albumin 
(Salomon).     It  has  also  been  found  in  the  spores  of  lyco- 


CHEMISTRY    OF    THE     LEUCOMAINES.  299 

podium,  and  in  the  pollen  of  various  plants,  in  seed  of  black 
pepper,  in  grass,  clover,  oats,  bran  of  wheat,  larvae  of  ants  ; 
in  the  juice  of  potato  (Schulze)  ;  in  certain  wines 
(Kayser)  ;  in  the  aqueous  decoction  of  yeast  of  beer 
(Schutzenberger)  ;  and  also  in  the  liquid  in  which  yeast 
is  grown  (Bechamp).  Demant  has  shown  it  to  be  rela- 
tively abundant  in  the  muscles  of  pigeons  in  a  state  of  in- 
anition, while  in  muscles  of  well-fed  pigeons  it  is  said  to 
be  entirely  absent.  Salomon  found  hypoxanthine  and 
xanthine  in  the  cotyledons  of  lupine,  as  well  as  in  the 
sprouts  of  malt,  while  Reinke  and  Rodewald  observed 
these  two  bases  together  with  guanine  in  .ZEthaliuni  sep- 
ticum — with  adenine,  xanthine,  and  theophylline,  it  occurs 
in  tea-leaves  (Kossel). 

Hypoxauthine  has  not  been  extracted  from  the  pancreas, 
where  it  seems  to  be  replaced  by  guanine,  or  rather  by 
adenine.  It  seems  that  hypoxanthine  bears  a  relation  to 
adenine  similar  to  that  which  we  see  between  glycocoll  and 
glycocollic  acid. 

Hypoxanthine  occurs  frequently  in  plants  together  with 
the  other  members  of  this  group,  namely,  adenine,  guanine, 
and  xanthine.  The  widely  distributed  character  of  these 
bases  is  due  to  the  presence  of  a  parent  substance,  viz., 
nuclein,  the  necessary  constituent  of  all  cells  capable  of 
development,  which  under  the  influence  of  acids,  and 
probably  likewise  of  ferments,  decomposes  into  the  above- 
mentioned  bases.  They  may,  therefore,  be  considered  as 
the  first  steps  in  the  retrograde  metamorphosis  of  all 
tissues,  since  they  have  their  origin  in  nuclein,  an  impor- 
tant proteid  substance.  Recent  advances  in  biological 
chemistry  have  shown  that  the  undeveloped  eggs  of  various 
insects  and  birds  yield  much  less  quantity  of  xanthine 
bodies  (hypoxanthine,  xanthine,  etc.)  on  treatment  with 
dilute  acid  than  the  partially  developed  eggs  (Tichomiroff, 
Kossel).  This  is  dependent  upon  the  remarkable  fact 
observed  by  Kossel  that  the  nuclein  of  undeveloped 
chicken  eggs  differs  from  the  nuclein  of  cell  nuclei  and 
resembles  that  obtained  from  milk.  For,  while  the  nuclein 
from   the   cell   nuclei   decomposes    into    adenine,  guanine, 


300  BACTERIAL    POISON'S. 

hypoxanthine,  etc.,  that  from  undeveloped  eggs  and  from 
milk  yields  no  nitrogenous  bases  on  treatment  with  acids. 
But  as  the  egg  develops,  i.e.,  the  nucleated  cells  increase  in 
number,  this  latter  nuclein  is  gradually  converted  or  gives 
way  to  the  ordinary  cell  nuclein,  and  hence  it  is  that  the 
chick  embryo  yields  guanine,  hypoxanthine,  and  possibly 
adenine. 

Unquestionably,  the  presence  of  hypoxanthine,  etc.,  in 
developing  cells  is  due  to  the  presence  of  the  nuclein  mole- 
cule, from  which  it  is  readily  split  off.  In  muscle,  however, 
hypoxanthine  and  xanthine  appear  to  exist  preformed,  and 
bear  no  relation  to  nuclein,  since  they  are  in  the  free  condi- 
tion, and  can  be  extracted  from  the  tissue  by  water.  For 
an  explanation  of  this  peculiar  fact,  see  Adenine,  page  284, 
and  Guanine,  page  308. 

According  to  the  observations  of  Salomon  and  Chit- 
tenden, hypoxanthine  is  formed  by  the  digestion  of  blood 
fibrin  with  gastric  juice,  pancreatic  juice,  or  on  heating  with 
water  or  dilute  acids.  Ew  albumin  under  the  same  con- 
ditions  does  not  yield  any  hypoxanthine,  except  when 
treated  with  pancreatic  juice.  These  observations  require 
repetition,  inasmuch  as  the  fibrin  used  undoubtedly  con- 
tained nuclein,  which,  as  we  now  know,  readily  decomposes 
under  those  conditions  into  its  characteristic  nitrogenous 
bases.  Be  that  as  it  may,  it  appears,  however,  to  be  one 
of  the  products  formed  by  the  decomposition  and  succes- 
sive oxidation  of  proteid  matter  previous  to  the  formation 
of  uric  acid  and  urea. 

When  a  mixture  of  guanine,  xanthine,  and  hypoxanthine 
is  allowed  to  putrefy,  the  bases  decompose  and  disappear  in 
the  order  named.  Hypoxanthine  resists  bacterial  action 
the  longest,  and  this  corresponds  with  its  behavior  to  re- 
agents (Baoinsky).  Adenine  during  putrefaction,  in  the 
absence  of  air,  is  converted  into  hypoxanthine,  and  guanine 
is  correspondingly  changed  into  xanthine  (Schindler). 
An  imido  group  is,  therefore,  replaced  by  oxygen,  and 
probably  goes  to  form  urea.  This  conversion  is  a  very 
important  fact,  since  the  process  of  putrefaction,  as  Hoppe- 
Seyler  has  repeatedly  pointed  out,  is  analogous  to  the 


CHEMISTRY    OF    THE     LEUCOM AINES.  301 

vital  process,  and  the  same  chemical  change  may  take  place 
in  the  animal  organs.  The  same  change  very  probably 
takes  place  in  the  auto-digestion  of  yeast.  Its  formation 
under  these  conditions  can  be  represented  thus  : 

C5H5N5  +  H20  =  C5H4N40  +  NH3. 

Hypoxanthine  can  be  readily  obtained  from  a  number  of 
closely  related  substances.  Thus,  carnine,  by  the  action  of 
oxidizing  agents,  is  converted  into  hypoxanthine  (page  328). 
For  this  reason  Weidel  and  Schutzenberger  regard 
hypoxanthine  as  derived  from  carnine,  but  this  view  is  now 
entirely  set  aside  by  our  present  knowledge  of  the  relation 
of  this  base  to  nuclein. 

Again,  it  can  be  obtained  from  adenine  (page  289)  by  the 
action  of  nitrous  acid.  The  relation  that  hypoxanthine 
bears  to  uric  acid  is  shown  by  the  fact  that  the  latter  is 
converted  by  nascent  hydrogen  first  into  xanthine,  and 
finally  into  hypoxanthine. 

C8H4N4Os  +  2H2  =  C5H4N40  +  2H20. 

Uric  Acid. 

This  transformation  of  uric  acid  into  hypoxanthine  is  of 
especial  importance,  since  together  with  Horbaczewski's 
synthesis  of  uric  acid,  accomplished  by  acting  on  urea  with 
either  glycocoll  or  trichlorlactamide,  it  constitutes  the  last 
step  in  the  complete  synthesis  of  hypoxanthine  from  its 
elements. 

Hypoxanthine  has  been  hitherto  regarded  as  a  step  lower 
than  guanine  in  the  series  of  nitrogenous  products  of 
regressive  metamorphosis,  and  consequently  was  considered 
as  derived  from  guanine.  The  investigations  of  Kossel, 
however,  show  that  it  arises  not  from  guanine  but  from 
adenine.  On  the  other  hand,  guanine  is  to  be  looked  upon 
as  the  source  of  xanthine.  It  is  probable  that  in  the 
organism  it  is  oxidized  as  soon  as  it  is  set  free  from  the 
nuclein,  forming  successively  xanthine,  uric  acid,  urea,  etc., 
and  the  small  quantity  present  in  the  urine  is  all  that  has 
escaped  oxidation.  When  fed  to  dogs,  it  was  observed  that 
the  amount  of  hypoxanthine  present  in  the  urine  decreased, 

14 


302  BACTEKIAL    POISON'S. 

and  even  became  less  in  amount  than  before  the  experiment ; 
but;  on  the  other  hand,  the  amount  of  xanthine  in  the  urine 
was  found  to  have  been  increased  above  the  normal.  This 
shows  that  hypoxanthine  in  the  body  is  oxidized  probably 
first  to  xanthine,  then  into  uric  acid.  According  to  Robert 
hypoxanthine  is  a  true  muscle  stimulant. 

The  fact  that  hypoxanthine  is  so  widely  distributed  in 
the  organism,  and  in  much  larger  quantities  than  was 
formerly  supposed,  shows  that  it  constitutes,  together  with 
the  closely  related  bodies  creatine,  xanthine,  guanine,  etc., 
the  normal  antecedents  of  urea  and  uric  acid.  This  view  is 
furthermore  strengthened  since  hypoxanthine  is  especially 
abundant  in  those  organs  which  are  most  active  m  pro- 
ducing metabolic  changes  in  the  body,  viz.,  the  liver  and 
spleen. 

It  may  be  prepared  from  the  urine,  according  to  the 
method  given  under  paraxanthine  (page  322) ;  or  from 
extract  of  meat,  or  from  glandular  organs,  such  as  the  liver, 
spleen,  etc.,  by  the  process  on  page  285.  Nuclein,  on  de- 
composition with  acids,  yields  about  one  per  cent,  of  this 
base.  It  can  be  determined  with  adenine  indirectly  by 
Schindler's  method  (page  286) ;  but  better  still  directly 
by  Bruhn's  picrate  method  (see  page  286).  After  the 
adenine  has  been  precipitated  by  sodium  picrate,  the  deter- 
mination of  hypoxanthine  in  the  filtrate  is  not  difficult  if 
hydrochloric  and  other  acids,  the  silver  salts  of  which  do 
not  quite  dissolve  in  ammonia,  are  absent.  The  filtrate 
from  the  adenine  picrate  is  rendered  slightly  alkaline  with 
ammonia  and  precipitated  with  silver  nitrate  at  the  boiling- 
point.  The  slightly  yellow-colored  precipitate  is  washed 
with  hot  water  till  the  wash-water  is  colorless  ;  then  dried 
at  120°  for  from  two  to  three  hours,  when  it  has  the  com- 
position 2C5H2Ag2N40  +  H20.  It  contains,  however,  traces 
of  picric  acid  and  some  adenine  silver,  and  hence  the  quan- 
tity of  hypoxanthine  calculated  from  the  weight  obtained  is 
higher  than  it  really  is.  Bruhns,  as  a  correction,  subtracts 
3.0  mg.  from  the  calculated  quantity  of  hypoxanthine. 

A  more  convenient  method  than  the  preceding  is  to  esti- 
mate hypoxanthine  as  hypoxanthine  silver  picrate.     The 


CHEMISTKY    OF    THE    LEUCOMAINES.         303 

filtrate  from  the  adenine  picrate  (page  287)  is  raised  to  the 
boiling-point~and  silver  nitrate  solution  gradually  added. 
The  precipitate  is  washed  with  cold  water  till  the  wash- 
water  is  colorless,  then  dried  at  100°,  when  its  composition 
is  represented  by  the  formula  C5H3AgN40.C6H2(NO2)3OH. 
The  calculated  quantity  of  hypoxanthine  here  is  likewise 
slightly  higher  than  it  should  be.  Bruhns  deducts  1.0 
mg.  from  the  calculated  result. 

In  the  presence  of  hydrochloric  acid,  etc.,  the  deter- 
mination of  hypoxanthine  is  somewhat  circuitous  since  the 
precipitated  silver  chloride  must  be  separated  from  the 
hypoxanthine  compound.  The  best  procedure  in  this  case 
is  to  saturate  the  filtrate  from  adenine  picrate  with  am- 
monia and  precipitate  it  completely  with  silver  nitrate. 
The  precipitate  is  washed  with  hot  water  (a  thorough  wash- 
ing is  not  necessary),  then  it  is  boiled  several  times  with 
nitric  acid  of  1.1  specific  gravity.  The  acid  each  time  is 
rapidly  decanted  on  to  a  small  filter,  and  finally  the  residue 
washed  on  the  filter  with  10  c.c.  of  the  hot  acid  (total  100 
c.c).  To  the  combined  acid  filtrate  silver  nitrate  is  added, 
and  the  whole  set  aside  for  twenty-four  hours.  The  pre- 
cipitate is  dried  at  100°.  The  amount  of  hypoxanthine 
lost  depends  upon  the  quantity  of  silver  chloride  present. 
The  correction  to  be  added  is  3.1  mg.  (Bruhns).  In 
Neubauer-Kossel's  method  the  mixed  adenine  and  hypo- 
xanthine silver  salts  can  be  decomposed  with  a  little  hydro- 
chloric acid  and  estimated  in  this  way. 

Hypoxanthine  is  a  white,  colorless,  crystalline  powder, 
sometimes  in  part  amorphous  ;  according  to  Bruhns,  pure 
hypoxanthine  does  not  form  floccules  and  bunches  of  micro- 
scopic needles,  but  usually  coherent  crusts,  which  consist  of 
roundish,  sharp -cornered  granules ;  some  resemble  quadratic 
octahedra.  It  is  soluble  in  about  300  parts  of  cold  water 
(Strecker).  The  base  separates  slowly  from  aqueous 
solutions,  and  when  pure  the  solubility,  even  in  the  begin- 
ning, is  less  than  1 :  300.  At  the  end  of  four  days  Bruhns 
found  it  to  be  1  :  1880.  It  is  more  easily  soluble  in  boiling 
water  (78  parts),  and,  on  cooling,  separates  in  the  form  of 
white,  crystalline  floccules,  thus  differing  from  xanthine, 


304  BACTERIAL    POISONS. 

which  is  amorphous.  The  solubility  in  cold  alcohol  is  very 
slight,  about  1  :  1000.  It  dissolves  in  acids  and  alkalies 
without  decomposition,  and  from  solutions  in  the  latter 
it  can  be  precipitated  by  passing  carbonic  acid,  or  by  the 
addition  of  acetic  acid.  The  aqueous  solution  possesses  a 
neutral  reaction.  The  free  base  can  be  heated  up  to  150° 
without  suffering  decomposition,  but  above  this  temperature 
it  sublimes,  and  partially  decomposes,  with  evolution  of 
hydrocyanic  acid.  When  heated  with  potassium  hydrate 
to  200°,  it  yields  ammonia  and  potassium  cyanide.  Heated 
with  water  to  200°,  it  decomposes  into  carbonic  acid,  formic 
acid,  and  ammonia,  and  in  this  respect  it  agrees  with  adenine 
(page  288).  The  properties  of  Strecker's  sarkine  agree 
closely  with  those  of  adenine-hypoxanthine  ;  and,  inasmuch 
as  the  latter  has  been  often  described  as  hypoxanthine,  it  is 
very  desirable  that  the  properties  of  hypoxanthine  be  re- 
determined. 

When  evaporated  with  an  oxidizing  agent,  chlorine  water 
or  nitric  acid,  the  residue  is  said  to  give  on  contact  with 
ammonia  vapors  a  rose-red  color  (Weidel,  murexide  test). 
Kossel,  however,  has  shown  that  this  is  due  to  the  presence 
of  xanthine,  and  that  pure  hypoxanthine  does  not  give  either 
the  murexide  test  or  the  xanthine  reaction.  According  to 
Strecker,  concentrated  nitric  acid  converts  hypoxanthine 
into  a  nitro-compound,  which  in  turn,  by  the  action  of  a 
reducing;  a»;ent,  is  changed  into  xanthine.  This  statement 
has  not  been  confirmed  either  by  Fischer  or  by  Kossel. 
It  does  not  give  a  green  color  with  sodium  hydrate  and 
chloride  of  lime — distinction  from  xanthine  (page  316). 

With  acids  it  yields  crystallizable  compounds,  and,  like 
the  amido  acids,  it  forms  compounds  with  bases,  and  also 
with  metallic  salts,  such  as  silver  nitrate  and  copper  acetate. 

The  hydrochloride,  C5H4N4O.HCl  +  H20,  crystallizes  in 
needles,  and,  like  the  nitrate  and  sulphate,  it  is  dissociated 
on  contact  with  water.  The  crystalline  form  is  character- 
istic and  distinct  from  that  of  adenine,  as  well  as  adenine- 
hypoxanthine.  The  nitrate  forms  thick  prisms  or  roundish 
masses,  readily  soluble  in  water  and  ammonia.     Platinum 


CHEMISTRY    OF    THE    LEUCOMAINES.  305 

chloride  forms  a  yellow,  crystalline  double  salt,  having  the 
composition  C5HXO-HCl.PtCl4. 

The  picrate  forms  yellow  prisms  easily  soluble  in  water, 
which  solution  is  not  affected  as  that  of  adenine  by  sodium 
picrate. 

Hypoxanthine  silver,  C5H2Ag2N4O.H20.  All  attempts 
to  obtain  a  compound  containing  but  one  atom  of  silver 
in  the  molecule,  corresponding  to  the  adenine  compound 
C5H4AgN5,  have  failed.  The  above  compound  was  first 
prepared  by  Strecker,  and  given  the  formula  C5H4N40. 
Ag20  ;  but  the  former  is  preferable,  since  on  heating  at  120° 
two  and  a  half  molecules  of  water  are  lost  and 

205H2Ag2]Sr4O  +  H20  (Ag  =  60.2  per  cent.)  results. 

At  140°-150°  it  loses  again  in  weight  and  becomes  gradu- 
ally gray ;  on  exposure  to  air  it  absorbs  moisture.  In  this 
form  hypoxanthine  can  be  estimated  quantitatively  (see 
page  302) ;  the  presence  of  sodium  picrate  does  not  interfere, 
but  chlorides,  etc.,  do.  It  is  insoluble  in  hot  water.  The 
compound,  C5H2Ag2N40.3H20,  is  obtained  in  the  form  of 
microscopic  needles,  by  treating  pure  hypoxanthine  silver 
nitrate  with  excess  of  aqueous  ammonia.  On  boiling  with 
ammonia-water  it  is  but  slightly  dissolved,  and  appears  to 
slowly  lose  a  part  of  its  water  of  crystallization.  As  a 
result  of  the  decomposition  one-half  of  the  hypoxanthine 
passes  into  solution  and  can  be  recovered  on  boiling  with 
addition  of  silver  nitrate  in  the  crystalline  form ;  or  in  the 
cold,  as  the  usual  amorphous  precipitate,  CsH2Ag2N4O.H20. 
Hypoxanthine  silver  nitrate,  C5H4N40. AgNOs,  (Ag  = 
35.29  per  cent.),  is  the  best-known  compound  ;  its  formula 
was  established  by  Strecker.  It  is  obtained  by  dissolving 
the  above  precipitate,  produced  by  addition  of  silver 
nitrate  to  an  ammoniacal  solution  of  the  base,  in  hot  nitric 
acid,  specific  gravity  1.1  ;  on  cooling  the  hypoxanthine 
silver  nitrate  crystallizes  in  the  form  of  tufts  of  microscopic 
needles  or  plates.  Heated  at  100°-120°  it  remains  con- 
stant in  weight ;  the  quantity  of  silver  present,  when  deter- 
mined, is  always  somewhat  higher  than  the  theoretical, 


306  BACTERIAL    POISONS. 

especially  if  an  excess  of  silver  nitrate  is  employed  in  the 
precipitation.  The  explanation  of  this  fact  is  probably 
that  given  under  Adenine,  though  presence  of  silver  chlo- 
ride may  partly  be  the  cause.  On  treatment  with  am- 
monia it  loses  not  only  nitric  acid  but  also  half  of  the 
hypoxanthine,  and  C5H2Ag2N40.3H20  forms.  The  change 
takes  place  readily  even  in  the  cold,  and  if  during  the 
digestion  an  excess  of  silver  nitrate  is  added,  the  hypo- 
xanthine set  free  is  converted  into  this  compound,  which  is 
wholly  constant  in  composition  compared  with  the  hypo- 
xanthine silver  nitrate.  The  conversion  is  quantitative. 
Very  dilute  hydrochloric  acid,  as  well  as  hydrogen  sulphide, 
removes  the  silver  from  this  compound. 
Hypoxanthine-  silver  picrate, 

C5H3AgN4O.C6H2(N02)3OH  (Ag  ==  22.88  per  cent.), 

is  gradually  formed  by  adding  silver  nitrate  to  a  boiling 
solution  of  hypoxanthine  picrate.  The  precipitate  is  granular 
and  of  a  lemon-yellow  color,  and  consists  of  aggregations  of 
fine  short  needles.  It  is  slightly  soluble  in  hot,  insoluble  in 
cold  water.  It  is,  therefore,  applicable  for  a  quantitative 
determination  of  the  base.  Aqueous  ammonia  very  readily 
and  completely  removes  the  picric  acid  from  the  compound, 
and  the  residue  is  hypoxanthine  silver,  which  is  slightly 
colored  yellow  by  a  trace  of  picric  acid  ;  half  of  the  hypo- 
xanthine passes  into  solution.  Nitric  acid  with  difficulty 
converts  it  into  hypoxanthine  silver  nitrate. 

Hypoxanthine  mercuric  chloride,  C5H3N4OHgCl,  is  ob- 
tained by  adding  an  equivalent  quantity  of  mercuric  chloride 
to  a  boiling  solution  of  hypoxanthine.  The  precipitate, 
which  increases  on  cooling,  is  crystalline. 

A  second  compound,  C5H3N4OHg2Cl3,  is  produced  by 
adding  a  strong  excess  of  mercuric  chloride,  in  the  cold,  to 
an  aqueous  solution  of  hypoxanthine.  It  forms  a  heavy 
granular  micro-crystalline  precipitate,  which  contains  some 
water  of  crystallization. 

By  boiling  the  preceding  compound  with  just  sufficient 
hydrochloric  acid  to  effect  complete  solution,  there  is  formed 
on  standing  a  precipitate  of  white  roundish  aggregates  of 


CHEMISTRY    OF    THE    LEUCOMAINES.  307 

leafy  or  needle-shaped  glittering  crystals  which  have  the 
composition  OsH4N4OHgCl2  +  H20. 

The  following  table  of  Bruhns  illustrates  the  analogy 
existing  between  the  mercury  compounds  of  adenine  and 
hypoxanthine  and  similar  derivatives  of  ammonium  : 

Ammonium.  Adenine.  Hypoxanthine. 

NH2HgCl  C5H4N5HgCl  C5H3N4OHgCl(+H20) 

NH2Hg2Cl3       C5H4N5H?2C13  C5H3N4OHg2Cl3(+H20) 

(NH3)2HgCl2{f§|^^2Cc^2(N02)30H  C5H4N4OHgCl2(+H.20) 

A  brom-hypoxanthine  compound  corresponding  to  that  of 
adenine  has  not  been  obtained. 

Benzyl-hypoxanthine,  C5H3N4O.CH2.C6H5,  was  obtained 
by  Thoiss  by  the  action  of  nitrous  acid  on  benzyl-adenine. 
It  forms  a  white  crystalline  mass  which  under  the  micro- 
scope consists  of  thin  plates.  It  is  easily  soluble  in  hot 
water,  dilute  alcohol,  and  in  acetic  ether ;  insoluble  in  ether 
and  chloroform.  It  melts  at  280°.  It  appears,  as  Kossel 
has  pointed  out,  that  adenine  and  hypoxanthine  contain  a 
group,  C5H4N4,  which  he  named  adenyl.  The  formation  of 
the  benzyl  derivatives  of  these  two  bases  shows  that  the 
hydrogen  atom  which  is  replaced  occurs  in  the  adenyl  and 
not  in  the  imido  group.  According  to  this  view  adenine  is 
to  be  considered  as  adenylimide  (C5H4N4.]SrH)  and  hypo- 
xanthine as  adenyloxide  (05H4N4O). 

Phosphomolybdic  acid  precipitates  hypoxanthine  from 
acid  solution,  and  in  general  it  gives  the  ordinary  alkaloidal 
reactions. 

It  is  not  precipitated  by  ammoniacal  basic  lead  acetate. 
Copper  acetate  does  not  precipitate  it  in  the  cold,  but  does 
on  boiling.  This  fact  has  been  made  use  of  in  the  isolation 
of  hypoxanthine.  Mercuric  chloride,  as  well  as  mercuric 
nitrate,  produces  a  flocculent  precipitate. 

Altogether,  in  its  behavior  to  reagents  it  resembles  xan- 
thine to  a  very  considerable  degree.  The  two  can  be 
separated,  however,  by  the  different  solubilities  of  the 
hydrochlorides  in  water,  and  more  especially  of  the  silver 
salt  in  nitric  acid. 

Physiological  Action. — 25-100  mg,  begin  to  act  on  frogs 


308  BACTEEIAL    POISONS. 

in  from  six  to  twenty-four  hours,  and  produce  increased  re- 
flex excitability  and  convulsive  attacks ;  5-100  mg.  is  fatal 
(Filehne).  When  injected  subcutaneously  into  hepato- 
tomized  geese  or  chickens  a  corresponding  increase  in  uric 
acid  secretion  is  observed  (v.  Mach).  This  conversion  is 
analogous  to  that  observed  by  Stadthagen  in  the  case  of 
guanine  (page  310),  and  shows  that  in  the  xanthine  bodies 
we  have  antecedents  of  uric  acid  apart  from  the  synthesis  of 
the  latter  from  ammonia  in  the  liver.  The  process  by  which 
this  change  is  effected  is  undoubtedly  one  of  oxidation. 

Guanine,  C5H5N50,  was  discovered,  in  1844,  by  Unger, 
as  a  constituent  of  guano,  in  which  it  is  present  in  varying 
quantities  according  to  the  region  from  which  the  guano 
comes.  Thus,  the  Peruvian  guano  is  reported  as  containing 
the  largest  proportion  of  this  base,  and  on  that  account  this 
variety  is  employed  when  it  is  desired  to  prepare  guanine. 
Since  its  discovery  by  Unger,  it  has  been  met  with  in  a 
very  large  number  of  tissues,  both  animal  and  vegetable ; 
in  the  liver,  pancreas,  lungs,  retina,  in  the  thymus  gland  of 
the  calf,  and  in  the  testicle  substance  of  the  bull ;  in  the 
scales  of  the  bleak,  and  in  the  swimming-bladder  of  fish,  as 
well  as  in  the  excrements  of  birds,  of  insects,  as  the  garden 
spider,  in  which  it  occurs  with  a  small  quantity  of  uric  acid 
(Weinmann),  and  is  to  be  regarded  as  a  decomposition 
product  of  proteicls  formed  in  the  tissues  of  the  spider.  It 
is  also  found  in  the  spawn  and  testicle  of  salmon,  and 
Schulze  and  others  have  shown  it  to  be  present  in  the 
young  leaves  of  the  plane-tree,  of  vine,  etc.,  also  in  grass, 
clover,  oats,  as  well  as  in  the  pollen  of  various  plants. 
Schutzenberger  has  isolated  it,  together  with  hypoxan- 
thine,  xanthine,  and  carnine,  from  yeast  which  had  been 
allowed  to  stand  in  contact  with  water  at  near  the  body- 
temperature.  Pathologically,  it  occurs  in  the  muscles,  liga- 
ments, and  joints  of  swine  suffering  from  the  disease  known 
as  guanine-gout.  Normally,  guanine,  like  adenine,  is 
present  in  muscle  tissue  only  in  traces.  It  has  never  been 
found  in  the  urine,  though  xanthine  has  been  mistaken  for 
guanine  by  some, 


CHEMISTRY    OF    THE    LEUCOMAINES.  309 

As  to  the  origin  of  this  sut stance  in  the  organism  very 
little  has  been  known  np  to  within  a  few  years,  except  so 
far  as  it  has  been  shown  to  be,  together  with  other  members 
of  this  group,  a  transitory  product  in  the  retrograde  meta- 
morphosis of  nitrogenous  foods  and  tissues.  In  the  case  of 
the  lower  animals  it  is  evidently  the  end-product  of  all 
change,  inasmuch  as  it  is  excreted  as  such.  Our  knowledge 
as  to  the  immediate  origin  of  this  and  the  other  allied  bases 
has  lately  been  extended  by  the  brilliant  researches  of 
Kossel  on  the  decomposition  products  of  nuclein,  in  which 
he  has  shown  that  this  essential  constituent  of  all  nucleated 
cells,  whether  animal  or  vegetable,  decomposes  under  the 
action  of  water  or  dilute  acids  into  adenine,  guanine,  hypo- 
xanthine,  and  xanthine.  We  know  that  the  first  two  bases 
are  readily  converted  by  the  action  of  nitrous  acid  into  the 
other  two ;  that  is  to  say,  an  NH  group  in  these  bases  is 
replaced  by  an  atom  of  0 — a  change  which  it  is  not  at  all 
unlikely  takes  place  in  the  tissues,  perhaps  in  every  cell 
nucleus.  That  such  a  change  is  quite  probable  is  shown  by 
the  putrefaction  experiments  of  Schindler,  whereby  aden- 
ine and  guanine  were  converted  respectively  into  hypoxan- 
thine  and  xanthine.  If  this  explanation  is  correct,  then 
adenine  and  guanine  are  transition-products  between  the 
complex  proteid  molecule  on  the  one  hand,  and  hypoxan- 
thine  and  xanthine  on  the  other.  These  two,  in  turn,  form 
the  connecting  link  to  the  last  step  in  the  regressive  meta- 
morphosis of  the  nitrogenous  elements  of  the  tissues,  viz., 
the  formation  of  uric  acid  and  urea.  We  can  thus  trace  a 
succession  of  cycles  from  the  complex  nuclein  molecule, 
which  is  apparently  indispensable  to  the  functional  activity 
of  all  reproductable  cells,  to  the  physiologically  waste  pro- 
ducts urea  and  uric  acid. 

Schtjlze  and  Bosshaed  recently  (1886)  found  in  young 
vetch,  clover,  ergot,  etc.,  a  new  base,  to  which  they  have 
given  the  name  vernine.  It  has  the  formula  C16H2nN808, 
and  is  of  especial  interest  at  this  point,  since  on  heating 
with  hydrochloric  acid  it  apparently  yields  guanine.  We 
have,  therefore,  at  least  two  well-defined  sources  of  guanine, 
the  nucleins  and  vernine. 

14* 


310  BACTERIAL    POISONS. 

Neither  adenine  nor  guanine  occur  in  normal  muscle 
further  than  in  mere  traces,  a  fact  which  can  only  be 
explained  on  the  ground  that  the  muscle  tissue  is  poor  in 
nucleated  cells,  and  hence  in  nuclein.  Just  as  the  muscle 
cell  has  become  morphologically  differentiated  from  the 
typical  cell,  it  may  be  looked  upon  also  as  having  under- 
gone a  concomitant  chemical  differentiation,  inasmuch  as  we 
no  longer  find  the  phosphoric  acid,  xanthine,  and  hypo- 
xanthine  in  the  same  chemical  combination  as  they  occur  in 
the  original  cell.  The  phosphoric  acid,  instead  of  existing 
as  a  part  of  an  organic  compound,  is  present  in  the  muscle 
tissue  as  a  salt ;  similarly  the  hypoxanthine  and  xanthine 
occur  in  the  free  condition,  extractable  by  water,  and  no 
longer  in  combination  Avith  other  groups  of  atoms  consti- 
tuting a  part  of  a  more  complex  molecule — nuclein. 

Guanine  and  creatine  apparently  mutually  replace  one 
another.  Thus,  in  the  muscle,  as  just  stated,  guanine  occurs 
only  in  traces,  whereas  creatine  is  especially  abundant. 
This  may  find  its  explanation  in  the  fact  that  both  are  sub- 
stituted guanidines.  Creatine  is  regarded  by  Hoppe- 
Seyler  as  an  intermediate  product  in  the  formation  of 
urea,  and  a  similar  role,  it  will  be  remembered,  belongs  to 
guanine.  From  Stadthagen's  experiments  on  dogs  we 
know  that  guanine  ingested,  produces  an  increase  in  the 
amount  of  uric  acid  and  urea  excreted,  aud  the  same  is 
also  true  of  the  nuclein  derived  from  yeast.  These  results 
have  led  him  to  the  conclusion  that  in  mammals  uric  acid 
is  a  direct,  or  more  or  less  altered  cleavage  product  of  pro- 
teids,  notwithstanding  the  fact  that  in  birds  it  is  the  result 
of  synthesis  in  the  liver. 

In  the  decomposition  of  nuclein-containing  substances, 
such  as  yeast,  liver,  spleen,  etc.,  by  dilute  acids,  neither 
adenine  nor  guanine  is  found  alone,  but  they  are  always 
accompanied  by  hypoxanthine,  and  usually  by  a  very  small 
quantity  of  xanthine. 

Guanine  may  be  readily  prepared  from  Peruvian  guano 
by  boiling  it  repeatedly  with  milk  of  lime  until  the  liquid 
becomes  colorless.  The  residue,  consisting  largely  of  uric 
acid  and  guanine,  is  boiled  with  a  solution  of  sodium  car- 


CHEMISTRY    OF    THE    LEUCOM  AINES.  311 

bonate,  filtered,  and  the  filtrate,  after  the  addition  of  sodium 
acetate,  is  strongly  acidulated  with  hydrochloric  acid.  This 
precipitates  the  guanine,  together  with  some  uric  acid.  The 
precipitate  is  dissolved  in  boiling  hydrochloric  acid,  and  the 
guanine  then  thrown  out  of  solution  by  the  addition  of  am- 
monium hydrate.  Guanine  is  also  obtained  in  the  decom- 
position of  nuclein  with  dilate  acids,  and  can,  therefore,  be 
prepared  from  such  cellular  organs  as  the  spleen,  pan- 
creas, etc.,  according  to  the  method  given  on  page  285.  It 
should  be  noted  here  that  in  the  decomposition  of  the  mixed 
silver  compounds  with  hydrogen  sulphide  or  ammonium 
sulphide  (Schindler)  the  guanine,  often  only  in  part,  passes 
into  solution  with  adenine  and  hypoxanthine,  and  the  re- 
mainder is  held  back  in  the  silver  sulphide  precipitate.  The 
latter  should,  therefore,  be  boiled  with  dilute  hydrochloric 
acid,  and  on  saturating  the  nitrate  with  ammonia  the  guan- 
ine after  a  while  separates.  That  portion  of  the  guanine 
which  did  pass  into  solution  with  the  other  two  bases  is 
separated  from  them  by  digestion  with  ammonia  on  a  water- 
bath.  The  two  portions  are  then  combined,  transferred  to 
a  filter,  previously  dried  at  110°  and  weighed,  washed  well 
with  ammonia,  then  dried  and  weighed. 

The  free  base  forms  a  white,  amorphous  powder,  insol- 
uble in  water,  alcohol,  ether,  and  ammonium  hydrate ; 
easily  soluble  in  mineral  acids,  fixed  alkalies,  and  in  excess 
of  concentrated  ammonium  hydrate.  It  can  be  heated  to 
above  200°  without  undergoing  decomposition.  When 
evaporated  with  strong  nitric  acid  it  gives  a  yellow  residue, 
and  this  on  the  addition  of  sodium  hydrate  assumes  a  red 
color,  which  on  heating  becomes  purple,  then  indigo-blue ; 
on  cooling  it  returns  to  a  yellow,  passing  through  purple 
and  reddish-yellow  shades  clue,  according  to  V.  Brucke,  to 
absorption  of  water.  This  is  the  so-called  xanthine  reac- 
tion, and  is  supposed  to  be  due  to  the  formation  of  xanthine 
and  a  nitro  product.  It  is  given  best  by  guanine,  then  by 
xanthine,  and  is  not  given  by  either  hypoxanthine  or 
adenine. 

Nitrous  acid  converts  it  directly  into  xanthine,  thus  : 

C5H5N50  +  HN02  =  G5H4N402  +  N2  +  H2Q. 


312  BACTERIAL    POISONS. 

This  reaction  is  identical  with  that  of  adenine,  whereby 
hypoxanthine  is  formed  (see  page  289).  By  putrefaction  in 
the  absence  of  air  it  forms  xanthine  (Schlndler).  The 
change  can  be  represented  by  the  equation  : 

05H5N5O  +  H20  =  C5H4]ST402  +  NH3. 

On  oxidation  with  potassium  permanganate  it  yields  urea, 
oxalic  acid,  and  oxy-guanine.  By  hydrochloric  acid  and 
potassium  chlorate  it  is  oxidized  to  carbonic  acid,  guani- 
dine,  and  parabanic  acid,  according  to  the  equation  : 

CO— NH  „  „ 

C5H5N50  +  H20  +  30  =  |  \  CO  +  M /C  =  NH  +  C0*' 

CO— NH7  a^7 

Parabanic  Acid.  Guanibine. 

According  to  Strecker,  a  small  amount  of  xanthine  is 
formed  in  this  reaction,  and  it  is  quite  possible  that  this 
base  is  also  formed  on  oxidation  with  nitric  acid. 

Guanine  combines  with  acids,  bases,  and  salts.  It  unites 
with  bases  to  form  crystalline  compounds  ;  and  with  one  or 
two  equivalents  of  acid  it  also  yields  crystallizable  salts. 
Thus,  with  hydrochloric  acid  it  forms  the  two  salts, 
08H6N5O.(HCl)2  and  C5H5N50.HC1  +  H20.  Similar  com- 
binations can  be  obtained  with  nitric  acid.  The  sulphate 
(C5H5N50)2H2S04,  crystallizes  in  long  needles,  and,  like 
the  other  salts,  is  decomposable  by  water.  The  platino- 
chloride,  (C5H,NsO.HCl)?PtCl4+  2H20,  is  readily  obtained 
in  a  crystalline  condition.  The  silver  compound  is 
soluble  in  hot  nitric  acid,  and  on  cooling  recrystallizes 
in  fine,  needle-shaped  crystals,  having  the  composition 
C5H5N50.AgNO3. 

The  solutions  of  the  hydrochloride  are  precipitated  by 
mercuric  chloride  and  nitrate,  potassium  chromate,  potas- 
sium ferricyanide,  and  by  picric  acid.  Basic  lead  acetate 
gives  a  precipitate  only  on  addition  of  ammonium  hydrate. 
The  reaction  with  picric  acid  (Capranica)  is  said  to  be 
very  characteristic,  and  a  means  of  distinguishing  this  base 
from  xanthine  and  hypoxanthine.  It  is  best  obtained  by 
adding  a  cold,  saturated  solution  of  picric  acid  to  the  warm 


CHEMISTRY    OF    THE    LEUCOMAINES.  313 

acidulated   solution  of  guanine,  when  a  light,  crystalline 

precipitate  forms.      Under  the  microscope   it  appears  in 

pencil-shaped,  fern-like  tufts  of  fine,  orange-yellow  needles. 

Physiologically  guanine  like  uric  acid  is  inert  (Filehne). 

Xanthine,  C5H4N402,  is  also  very  widely  distributed  in 
the  organism,  and  has  been  met  with  in  almost  all  the 
tissues  and  liquids  of  the  animal  economy.  Together  with 
hypoxanthine,  guanine,  and  possibly  adenine,  it  occurs  in 
many  plants,  among  which  may  be  mentioned  lupine, 
sethalium,  sprouts  of  malt,  tea-leaves  (Baginsky),  auto- 
digestion  of  yeast,  gourd  seeds,  soja  beans,  etc.  It  was 
first  discovered  by  Maecet  (1819)  in  a  urinary  calculus, 
and  since  then  has  been  frequently  found  as  the  only  or 
chief  constituent  of  many  calculi.  Unger  and  Phipson 
have  extracted  it  from  guano,  while  Salomon  has  shown 
it  to  be  one  of  the  products  formed  in  the  pancreatic  diges- 
tion of  fibrin.  Schutzenberger  found  it  together  with 
carnine  and  hypoxanthine  in  the  liquors  from  yeast.  It  is 
a  normal  constituent  of  the  urine,  but  is  present  only  in 
extremely  minute  quantities.  During  the  use  of  sulphur- 
baths,  or  after  the  thorough  application  of  sulphur  salves, 
the  quantity  of  xanthine  in  the  urine  is  considerably  in- 
creased. It  is  likewise  more  abundant  in  the  urine  of  leuco- 
cythsemic  patients,  for  the  reasons  already  given  on  page 
283.  Baginski  holds  that  the  amount  of  xanthine  nor- 
mally present  in  the  urine  may  be  increased  tenfold  in  the 
case  of  acute  nephritis.  Bence  Jones  observed  in  the 
urine  of  a  child  sick  with  renal  colic,  a  deposit  of  crystals 
which  he  considered  to  be  xanthine,  but  other  observers 
are  inclined  to  regard  the  crystals  as  those  of  hypo- 
xanthine. Vaughan  has  reported  the  presence  of  xan- 
thine in  deposits  from  the  urine  of  patients  with  enlarged 
spleen. 

Xanthine  may  be  prepared  synthetically  in  several  ways. 
Thus,  it  may  be  obtained  by  the  reduction  of  uric  acid  by 
means  of  sodium  amalgam,  according  to  the  equation  : 

C5H4N403  +  H2  =  C5H4N402  +  H20. 

ITeic  Acid.  Xanthine. 


314  BACTEKIAL    POISONS. 

ISTow  that  uric  acid  has  been  prepared  synthetically,  this 
forms  the  final  step  in  the  complete  synthesis  of  xanthine. 
By  further  action  of  nascent  hydrogen  the  xanthine  in  turn 
is  converted  into  hypoxanthine.  The  reverse  operation, 
the  conversion  of  hypoxanthine  into  xanthine,  though  re- 
ported by  Strecker  has  not  been  confirmed  by  Fischer 
or  by  Kossel.  It  is,  therefore,  evident  that  these  bodies 
form  a  continuous  oxidation  series  with  uric  acid  as  the 
final  product.  Although  this  change  is  unquestionably  the 
one  which  goes  on  in  the  animal  economy,  yet  all  attempts 
to  reproduce  it  in  the  laboratory  by  oxidation  with  potas- 
sium permanganate  or  nitric  acid  have  apparently  yielded 
only  negative  results.  Again,  xanthine  may  be  prepared 
from  guanine  by  putrefaction  of  the  latter,  or  by  oxidation 
with  nitrous  acid.  The  change  may  be  represented  by  this 
equation  : 

C5H5N50  +  HN02  =  C5H4N402  +  N2  +  H20. 

Guanine.  Xanthine. 

This  reaction,  first  described  by  Strecker  (1858),  corre- 
sponds exactly  to  the  one  by  which  Kossel  has  transformed 
adenine  into  hypoxanthine  (see  page  289). 

Gautier,  starting  out  on  the  hypothesis  that  xanthine 
is  a  polymerization-product  of  hydrocyanic  acid,  has  en- 
deavored to  prepare  it  directly  from  this  compound.  In- 
deed, he  claims  to  have  succeeded  in  effecting  the  synthesis 
of  not  only  xanthine,  but  also  its  homologue,  by  simply 
heating  hydrocyanic  acid  in  a  sealed  tube  with  water  and  a 
little  acetic  acid,  the  latter  being  added  to  neutralize  any 
ammonia  that  might  form.  He  expresses  the  reaction  as 
follows  : 

11HCN  +  4H20  =  C5H4N402  +  C6H6N402  +  3NH3. 

Xanthine.  Methyl-xanthine. 

Nearly  all  of  the  methods  that  have  been  employed  for 
the  preparation  of  xanthine  are  based  upon  its  precipitation 
as  the  insoluble  silver  compound.  From  the  urine  it  can 
be  isolated  according  to  the  method  given  under  paraxan- 
thine,  on  page  322.      It  may  also  be  obtained  from  the 


CHEMISTRY    OF    THE    LEUCOM AINES .  315 

urine  by  Hofmeister's  method.  The  urine,  acidulated 
with  hydrochloric  acid,  is  precipitated  with  phosphotungstic 
acid ;  the  precipitate  is  decomposed  by  warming  with 
baryta,  filtered,  and  the  filtrate  is  freed  from  barium  by  the 
cautious  addition  of  sulphuric  acid.  The  solution  is  then 
made  alkaline  with  ammonium  hydrate,  any  traces  of  phos- 
phates that  appear  are  filtered  off,  and  finally  it  is  precipi- 
tated by  addition  of  ammoniacal  silver  nitrate.  The  pre- 
cipitate which  forms  consists  of  the  silver  compounds  of  the 
xanthine  bodies,  and  is  purified  by  dissolving  in  hot  nitric 
acid,  as  given  on  page  285.  Xanthine  has  been  shown  to 
be  formed  at  the  same  time  with  guanine,  adenine,  and 
hypoxanthine,  in  the  decomposition  of  nuclein  by  means  of 
dilute  acids.  It  may,  therefore,  be  prepared  from  cellular 
organs  according  to  the  method  given  under  Adenine.  The 
method  of  its  preparation  from  tea-leaves  is  also  given 
elsewhere. 

Xanthine  is  a  white,  granular,  amorphous  body,  and  is 
deposited  from  hot  aqueous  solution  on  cooling  in  colorless 
floccules,  or  as  a  fine  powder,  which,  under  the  microscope, 
is  seen  to  consist  of  rounded  granules.  When  occurring  in 
calculi,  it  forms  compact,  moderately  hard,  yellow  or  brown 
fragments,  which,  on  being  rubbed  with  the  finger-nail, 
assume  a  wax-like  appearance.  It  is  difficultly  soluble  in 
cold  water  (about  14,000  parts),  alcohol,  and  ether ;  some- 
what more  soluble  in  boiling  water  (about  1200  parts).  It 
is  soluble  in  alkalies  and  alkali  carbonates,  not  bicarbon- 
ate, and  from  these  solutions  it  is  precipitated  on  neutral- 
ization with  acids,  or  by  passing  carbonic  acid.  In  warm 
ammonia  it  dissolves  more  readily  than  does  uric  acid  or 
guanine,  and  on  cooling  the  ammonium  compound  recrys- 
tallizes.  It  acts  as  a  weak  base,  and  as  a  weak  acid  ;  with 
salts  of  the  heavy  metals  it  forms  difficultly  soluble  or 
insoluble  compounds.  Its  basic  properties,  however,  are 
weaker  than  those  of  liypoxanthine  or  guanine. 

When  xanthine  is  evaporated  with  nitric  acid  it  leaves  a 
lemon-yellow  residue  (hence  its  name),  which  is  not  changed 
by  ammonium  hydrate — distinction  from  uric  acid — but 
with  potassium  hydrate  becomes  yellowish-red,  on  heating 
purple-red.    When  added  to  a  mixture  of  bleaching  powder 


316  BACTERIAL    POISONS. 

and  sodium  hydrate  in  a  watch-glass  the  solution  becomes 
covered  by  a  dark-green  scum,  which  changes  to  a  brown, 
and  soon  disappears — distinction  from  hypoxanthine. 

By  means  of  a  very  interesting  synthetic  reaction,  xan- 
thine may  be  converted  into  theobromine,  the  active  con- 
stituent of  Theobroma  cacao.  Thus,  the  xanthine  is  dissolved 
in  a  sufficient  quantity  of  sodium  hydrate,  necessary  to  form 
the  neutral  compound  C5H2BTa2N402,  and  this  product, 
when  treated  with  boiling  acetate  of  lead,  yields  a  white 
precipitate  of  lead  xanthine,  C5H2PbN402.  This  is  dried 
at  130°,  then  heated  for  twelve  hours  at  100°  with  methyl 
iodide,  when  the  dimethyl  derivative,  C5H2(CH3)2N402,  is 
formed.  This  compound  is  identical  with  the  natural  theo- 
bromine, and  by  a  similar  treatment  is  converted  into  tri- 
methyl- xanthine  or  caffeine.  The  relation  of  xanthine  to 
theine  (caffeine)  is  shown  in  the  fact  that  it  exists  together 
with  hypoxanthine,  adenine,  and  possibly  guanine,  in  fresh 
tea-leaves.  It  is,  therefore,  clear,  that-  by  starting  from 
guanine  of  guano  we  can  produce  successively  xanthine, 
dimethyl  xanthine,  and  trimethyl  xanthine,  the  last  two 
compounds  being  identical  with  the  alkaloids  of  theobroma 
and  of  coffee. 

Nascent  hydrogen  converts  this  base  into  hypoxanthine, 
but  the  reverse  operation,  the  oxidation  of  hypoxanthine 
into  xanthine,  has  been  questioned  of  late  by  Kossel  and 
others.  On  heating,  a  small  portion  volatilizes;  the 
greater  part  decomposes  into  ammonium  carbonate,  cyan- 
ogen, and  hydrocyanic  acid.  Heated  to  200°  with  hydro- 
chloric acid,  it  decomposes  with  the  formation  of  ammonia, 
carbonic  acid,  formic  acid,  and  glycocoll  (E.  Schmidt). 
When  bromine  is  allowed  to  act  on  xanthine,  there  is 
formed  a  substitution  compound,  having  the  formula 
C6H3Bi\N"402.  With  potassium  chlorate  and  hydrochloric 
acid  it  yields  alloxan  and  urea. 

Xanthine  is  a  weak  base,  which  dissolves  in  acids  with 
the  formation  of  salts. 

The  hydrochloride,  C5H4N402.HC1,  is  difficultly  soluble 
in  water,  more  so  than  the  corresponding  salt  of  hypoxan- 
thine, from  which  it  is  deposited  in  glistening   six-sided 


CHEMISTRY    OF    THE    LEUCOM  AINES.  317 

plates,  often  forming  aggregations.  Its  solution  does  not 
precipitate  platinum  chloride.  The  nitrate  forms  fine  yellow 
crystals. 

The  sulphate,  C,H4:N"402.II2S04  +  H20,  crystallizes  in 
microscopic  glistening  rhombic  plates,  decomposable  by 
water. 

With  baryta  water  xanthine  forms  the  difficultly  soluble 
compound  C5H4N402.Ba(OH)2;,  which  corresponds  to  the 
hypoxanthine  salt  C5H4lSr4O.Ba(OH)2,  and  to  that  of 
guanine. 

From  ammoniacal  solution,  silver  nitrate  precipitates  the 
compound  C5H41S"402.  Ag20,  which  is  insoluble  in  ammonia, 
but  soluble  in  hot  nitric  acid.  From  the  nitric  acid  solu- 
tion, on  long  standing,  there  separates  the  compound 
C5H4N402.AgN03,  which,  on  contact  with  water,  decom- 
poses, giving  off  nitric  acid.  The  ammoniacal  solution  is 
also  precipitated  by  lead  acetate — separation  from  hypo- 
xanthine— also  by  calcium  and  zinc  chlorides.  Cupric 
acetate  gives  a  precipitate  only  on  boiling.  The  aqueous 
solution  is  not  precipitated  by  lead  acetate,  but  is  by  phos- 
phomolybdic  acid,  phosphotungstic  acid,  by  mercurous  and 
mercuric  salts.  Picric  acid  gives  an  easily  soluble  com- 
pound, which  resembles  that  of  hypoxanthine,  but  differs 
from  that  of  guanine. 

As  to  the  physiological  relation  of  xanthine  very  little 
need  be  said.  It  bears  the  same  relation  to  guanine  that 
hypoxanthine  does  to  adenine,  and,  like  the  latter,  is  to  be 
looked  upon  as  an  intermediate  compound,  a  step  lower 
than  guanine,  and  nearer  the  limit  of  oxidation — uric  acid. 
It  is  quite  probable  that  in  the  body  it  is  oxidized  about 
as  rapidly  as  it  is  formed.  Like  hypoxanthine,  it  is  to 
be  regarded  as  a  true  muscle  stimulant,  especially  of  the 
heart.  (Bag-inski).  According  to  Filehne  it  produces 
in  frogs  a  decided  muscular  rigor  and  paralysis  of  the  spinal 
cord.  The  heart  muscle  is  also  affected,  which  is  not  the 
case  with  caffeine  or  theobromine.  The  fatal  dose  is  less 
than  one-half  pro  mille.  In  its  action  it  is  stronger  than 
theobromine,  while  caffeine  is  weaker  than  either  of  the 
two.     Paschkis  and  Pal  hold  that  the  reverse  is  true. 


318  BACTEKIAL    POISONS. 

In  closing  the  description  of  the  preceding  bodies  it  may 
be  well  to  present  briefly  our  present  knowledge  as  to  their 
constitution.  Gautier,  starting  out  with  the  idea  that 
they  are  polymerization-products  of  hydrocyanic  acid,  has 
deduced  theoretically  cyclic  formulae,  recalling  the  hexagon 
of  the  benzole  derivatives.  These  formulae,  though  formid- 
able in  appearance,  are  a  complete  failure  so  far  as  they  are 
expressions  of  chemical  reactions.  Thus,  the  formula  of 
guanine : 

H—  CO  —  N\„      ^>C  =  NH 


N  = 

=  CH 

N<c- 

-o>c  = 

/? 

% 

HN 

NH 

fails  to  show  either  a  urea  or  a  guanidine  residue,  and  yet 
it  is  a  well-known  fact  that  guanine  on  oxidation  yields 
parabanic  acid  and  guanidine  (page  312).  In  a  similar 
manner,  his  xanthine  formula  fails  to  show  up  the  urea 
residues  which  we  know  to  be  present. 

Hoebaczewski's  synthesis  of  uric  acid  has  thrown  con- 
siderable light  upon  the  constitution  of  these  bases.  As  a 
consequence  of  his  method  of  synthesis  uric  acid  was  shown 
to  possess  the  structural  formula  given  below.  E.  Fischer 
has  found,  as  a  result  of  experimental  work,  the  constitu- 
tion of  xanthine  to  be  expressed  by  the  subjoined  formula. 
We  know  that  uric  acid  on  treatment  with  nascent  hydro- 
gen is  converted  into  xanthine,  then  into  hypoxanthine. 
It  follows,  therefore,  that  a  relation  exists  between  hypo- 
xanthine and  xanthine  similar  to  that  between  xanthine 
and  uric  acid.  The  formula  of  hypoxanthine,  as  deduced 
from  this  relation,  and  given  below,  probably  represents 
its  constitution  quite  closely.  It  is  possible,  however,  that 
the  CH  and  CO  groups  will  be  found  to  occupy  the 
reverse  position  which  they  are  given  in  this  formula,  in 
which  case  corresponding  changes  must  be  made  in  the 
formulae  of  guanine  and  adenine.  The  latter  two  are  based 
upon  the  relation  which  these  bodies  bear  to  xanthine  and 


CHEMISTEY    OF    THE     LEUCO  M  AINES  , 


319 


hypoxanthine,  and  cannot  be  said  to  be  the  result  of  direct 
experimental  evidence. 


NH— C  —  NH 

I 
CO 

I 
NH— C       CO 

I  I 

CO— NH 

Uiuc  Acid. 

C5H4N403 


N  =  C  — NH 

I 
CO 

I 
NH— C       CO 

II         I 
CH— NH 

Xanthine. 

C5H4N402 


N  =  C  —  NH  N=C 

I  I 

CO  CH 

I  II 

NH— C       C=NH  N— C 

II  I 

CH— NH 


N=C  —  NH 

I 
CH 

II 
N— C       CO 

II        I 
CH— NH 

Hypoxanthine. 

C5H4N40 
-  NH 

C=NH 


C5H5N50 
Heteroxanthine,  C6H6N40, 


CH— NH 

Adenine.. 

C5H5N5- 


_  is  a  new  base  which  was 
isolated  from  the  urine  in  1884  by  Salomon.  In  its 
composition  it  is  methyl-xanthine,  and  is  intermediate 
between  xanthine  and  paraxanthine  or  dimethyl-xanthine. 
It  occurs  in  the  urine  of  man  and  of  the  dog  in  about  the 
same  amount  as  paraxanthine,  and  the  method  for  its  isola- 
tion will  be  found  under  the  description  of  that  base.  It 
is  a  remarkable  fact  that  this  base  occurs  in  dog's  urine 
unaccompanied  by  paraxanthine,  and  the  same  seems  to 
hold  true  for  the  urine  of  leucocythsemic  persons.  Salomon 
examined  the  liver  and  muscles  of  a  dog,  but  was  unable 
to  obtain  any  heteroxanthine  or  paraxanthine,  and  the  total 
amount  of  xanthine  bodies  present  was  about  normal. 
Hence,  he  is  inclined  to  think  that  these  two  bases  may 
possibly  have  their  origin  in  the  kidney.  Unlike  the  other 
xanthine  bodies,  heteroxanthine  has  not  as  yet  been  isolated 


320  BACTERIAL    POISONS. 

from  plants,  meat  extract,  or  guano.  The  amount  of 
xanthine  bodies  present  in  the  urine  is  unaffected  by  phos- 
phorus poisoning.  Neither  this  base  nor  paraxanthine  has 
been  found  in  bull's  testicles  ;  xanthine  is  also  absent,  and 
only  hypoxanthine  and  guanine  were  found  to  be  present. 

Heteroxanthine  forms  a  white  amorphous  powder,  which 
sometimes  on  prolonged  contact  with  water  forms  micro- 
scopic crystalline  tufts.  It  is  very  difficultly  soluble  in 
cold  water ;  much  more  easily  in  hot  water,  and  the  solu- 
tion thus  obtained  is  neutral  in  reaction.  It  is  easily  soluble 
in  ammonium  hydrate,  but  is  insoluble  in  alcohol  and  ether. 
When  heated  it  volatilizes  without  melting  and  at  the  same 
time  gives  off  a  small  quantity  of  hydrocyanic  acid.  On 
evaporation  with  nitric  acid  on  the  water-bath  (xanthine 
reaction)  it  remains  as  a  pure  white  residue,  which  on  con- 
tact with  sodium  hydrate  develops  only  a  trace  of  reddish 
coloration  or  none  at  all.  Weidel's  test  (page  328)  pro- 
duces a  splendid  red  color,  which  becomes  blue  on  the  ad- 
dition of  sodium  hydrate.  Simple  evaporation  with  chlo- 
rine water  gives  a  similar  though  not  so  strong  a  color 
reaction. 

Silver  nitrate  produces  in  ammoniacal,  as  well  as  in 
nitric  acid  solutions,  a  precipitate  which  readily  dissolves 
on  warming  in  even  very  dilute  nitric  acid ;  from  this 
solution,  if  not  too  concentrated,  the  heteroxanthine  silver 
nitrate  compound  crystallizes  in  well-formed  plate-like 
prismatic  crystals.  Copper  acetate  produces  in  the  cold,  in 
solutions  of  heteroxanthine,  a  clear-green  precipitate.  It 
is  also  precipitated  by  phosphotungstic  acid,  and  by  ammo- 
niacal basic  lead  acetate.  Picric  acid  does  not  give  a  yellow- 
colored  precipitate  in  solutions  of  the  hydrochloride.  Mer- 
curic chloride  readily  precipitates  heteroxanthine  in  the 
form  of  a  grayish-yellow  compound,  which  on  standing 
twelve  to  twenty-four  hours  becomes  converted  into  pure 
white  crystalline  aggregations.  This  mercuric  compound 
can  be  converted  directly  into  the  corresponding  silver 
compound  by  the  addition  of  silver  nitrate  and  ammonia, 
as  described  under  paraxanthine. 

The  hydrochloride  is  characterized  by  its  rather  difficult 


CHEMISTRY    OF    THE    LEUCOM AINES.  321 

solubility  and  ready  crystallization  (a  distinction  from  the 
paraxanthine  salt).  The  salt  forms  large  colorless  tufts 
of  crystals,  which  on  contact  with  water  soon  lose  their 
transparency  and  become  opaque  ;  gradually  their  crystal- 
line form  disappears,  till  nually  they  completely  decom- 
pose with  the  formation  of  heteroxanthine.  This  decom- 
position is  hastened  by  warming,  either  with  or  without 
addition  of  ammonia.  Platinum  chloride  produces  in  the 
hydrochloric  acid  solution  a  precipitate  of  crystalline  double 
salt. 

This  base  resembles  paraxanthine  in  its  property  of 
yielding  a  difficultly  soluble  precipitate  with  the  fixed 
alkali.  This  reaction  is  best  brought  about  by  dissolving 
the  heteroxanthine  hydrochloride  in  warm  dilute  sodium 
hydrate,  when,  on  cooling,  the  corresponding  sodium  salt 
will  crystallize  out  in  oblique-angled  plates.  These  crystals 
dissolve  easily  in  water,  and  on  neutralization  of  the 
solution  with  an  acid  a  dense  pulverulent  precipitate  of 
heteroxanthine  forms.  It  can  thus  be  distinguished  from 
paraxanthine,  the  sodium  compound  of  which,  on  similar 
treatment,  yields  the  characteristic  crystalline  form  of  the 
free  base.  This  sodium  reaction,  therefore,  distinguishes  it 
at  once  from  xanthine,  hypoxanthine,  guanine,  and  para- 
xanthine. It  differs  from  the  latter,  as  has  already  been 
indicated,  in  the  solubility  and  amorphous  character  of  the 
free  base ;  in  the  behavior  of  the  hydrochloride  and  the 
sodium  compound,  and  in  the  not  giving  a  precipitate  with 
picric  acid,  nor  the  characteristic  odor  given  by  paraxan- 
thine on  heating. 

In  its  composition,  heteroxanthine  is,  as  has  already 
been  stated,  methyl-xanthine  and  probably  is  related  to  if 
not  identical  with  an  isomeric  body  obtained  synthetically 
by  Gautier  (see  page  314).  The  fact  nevertheless  re- 
mains, that  in  the  urine  we  have  normally  a  homologous 
series  of  xanthine  bodies,  namely,  xanthine,  heteroxanthine, 
and  paraxanthine. 

Paraxanthine,  (^Ey^O^  was  isolated  in  1883  by 
Salomon,  who  has  since  shown  it  to  be  a  constituent  of 


322  BACTERIAL    POISONS. 

normal  urine,  although  present  in  exceedingly  minute 
quantity.  Thus  from  1200  litres  of  urine,  only  1.2  grammes 
(0.0001  per  cent.)  of  this  substance  were  obtained.  It  has 
not  been  found  in  the  urine  of  dogs  or  in  that  of  leuco- 
cythsemic  patients.  Thudichum  was  the  first  to  isolate 
paraxanthine  from  the  urine,  and  he  named  it  urotheo- 
bromine  (1879). 

The  method  employed  for  the  isolation  of  this  base  is, 
with  a  slight  modification,  that  of  E.  Salkowski,  as 
originally  proposed  for  the  preparation  of  xanthine  bases 
from  urine.  The  urine  in  portions  of  25  to  50  litres  is 
made  alkaline  with  ammonium  hydrate  and  allowed  to 
stand  twenty-four  hours.  The  clear  supernatant  fluid  is 
decanted  from  the  precipitate  of  phosphates  and  treated 
with  silver  nitrate  (0.5  to  0.6  gramme  per  litre).  The  gray- 
ish precipitate  of  xanthine  compounds  which  forms  is  trans- 
ferred to  a  filter  and  washed  with  water  till  free  from 
chloride ;  it  is  then  suspended  in  water  and  decomposed 
with  a  current  of  hydrogen  sulphide.  The  liquid  is  filtered 
by  decantatiou  and  the  filtrate  is  evaporated  to  dryness ; 
the  residue  is  extracted  with  3  per  cent,  sulphuric  acid  to 
remove  uric  acid ;  the  solution  thus  obtained,  after  it  has 
been  rendered  alkaline  with  ammonia,  is  precipitated  by 
silver  nitrate. 

A  better  procedure  is  to  concentrate  the  filtrate  directly 
over  the  flame  or  on  the  water-bath,  till  the  uric  acid  begins 
to  crystallize  out.  It  is  then  filtered,  and  the  filtrate,  after 
diluting  somewhat  with  water,  is  rendered  alkaline  with 
ammonium  hydrate  in  order  to  precipitate  any  remaining 
uric  acid  and  phosphates.  The  whole  is  allowed  to  stand 
one  or  two  days,  then  filtered,  and  the  filtrate  again  pre- 
cipitated with  silver  nitrate.  The  thoroughly  washed  pre- 
cipitate of  the  xanthine  compounds,  now  free  from  uric 
acid,  is  dissolved  in  as  little  as  possible  of  hot  nitric  acid 
of  specific  gravity  1.1,  to  which  a  little  urea  has  been 
added,  and  the  clear  solution  is  set  aside  for  twenty-four 
hours.  The  silver  salt  of  hypoxanthine  crystallizes  from 
the  solution  and  is  filtered  off.  It  can  be  purified  by  re- 
peated recrystallization  from  hot  nitric  acid,  containing  a 


CHEMISTRY    OF    THE    LEU COM AINES .  323 

little  urea,  then  decomposed  with  hydrogen  sulphide,  and 
the  nitrate,  rendered  alkaline  with  ammonium  hydrate,  is 
concentrated  to  a  small  volume.  On  standing,  pure  hypo- 
xanthine  crystallizes  out.  The  filtrate  from  the  silver  salt 
of  hypoxanthine  on  being  rendered  alkaline  with  ammonium 
hydrate  gives  a  precipitate  which  formerly  was  regarded  as 
consisting  entirely  of  the  xanthine  silver  compound,  but 
which  from  the  investigations  of  Salomon,  has  been  shown 
to  be  a  mixture  of  the  salts  of  xanthine,  paraxanthine, 
and  heteroxanthine. 

The  separation  of  these  bases  is  effected  by  the  solubility 
of  the  free  bases  in  ammonium  hydrate.  For  this  purpose 
the  precipitate  of  the  mixed  silver  salts  is  decomposed  with 
hydrogen  sulphide,  and  the  filtrate,  rendered  ammoniacal  to 
remove  traces  of  phosphates  and  oxalates,  is  moderately 
concentrated.  After  standing  twenty-four  hours,  heteroxan- 
thine crystallizes  out,  partly  in  finely  formed  sheaves  and 
tufts  of  needles,  partly  in  radially  striated  masses.  The 
fluid  is  decanted  from  the  crust  of  heteroxanthine  which 
forms  in  the  bottom  of  the  beaker,  and  after  being  concen- 
trated somewhat  is  again  allowed  to  stand.  In  this  way  a 
second  crop  is  obtained,  and  this  is  repeated  till  finally  the 
separated  masses  scarcely  give  a  precipitate  with  sodium 
hydrate.  All  the  heteroxanthine  is  now  united  and  dis- 
solved in  a  little  hot  water  by  the  aid  of  sodium  hydrate. 
After  twenty-four  hours  the  greater  part  of  the  heteroxan- 
thine crystallizes  out  in  bunches  of  crystals  of  sodium 
heteroxanthine,  while  a  small  part  together  with  any  traces 
of  xanthine  remains  in  solution.  The  crystalline  mass  is 
dried  by  pressure,  dissolved  in  a  little  water,  and  the  solu- 
tion neutralized  by  addition  of  hydrochloric  acid,  when  the 
heteroxanthine  separates  as  a  pulverulent  precipitate.  To 
remove  any  traces  of  paraxanthine,  dissolve  in  hydrochloric 
acid ;  on  standing  forty-eight  hours  the  heteroxanthine  salt 
separates,  while  the  easily  soluble  salt  of  paraxanthine 
remains  in  solution.  To  obtain  the  pure  free  heteroxan- 
thine, the  hydrochloric  salt  is  evaporated  with  ammonium 
hydrate ;  the  well-washed  residue  of  heteroxanthine  is  then 
dissolved  in  dilute  ammonia,  the  solution  filtered,  evapor- 


324  BACTEEIAL    POISONS. 

ated  slowly,  and  the  precipitate  which  forms  is  finally  washed 
with  alcohol  and  ether. 

The  original  ammoniacal  mother-liquors  of  heteroxan- 
thine  yield  on  further  concentration  amorphous  floccules  of 
xanthine,  which  are  removed  by  filtration ;  from  the  nitrate, 
when  concentrated  still  more,  paraxanthine  crystallizes  out. 

Paraxanthine  is  obtained  in  colorless,  glassy,  generally 
six-sided  plates,  which  are  arranged  in  tufts  or  rosettes. 
From  very  concentrated  aqueous  solutions  it  crystallizes  in 
long,  colorless,  interwoven  needles,  which  on  drying  exhibit 
the  silky  lustre  of  tyrosin.  The  crystals  belong  to  the 
monoclinic  system,  and  may  crystallize  with  as  well  as 
without  water.  If  water  is  present  on  careful  heating  (110°) 
the  crystals  lose  their  brilliancy  and  become  whitish  and 
opaque,  and  at  120°-130°  the  water  is  completely  driven 
off.  The  conditions  under  which  crystals  containing  water 
are  formed  are  not  known ;  probably  by  slow  crystalliza- 
tion, whereas  rapid  crystallization  from  hot  concentrated 
solution  yields  the  anhydrous  needles.  At  about  170°-180° 
sublimation  takes  place.  The  melting-point  is  at  about 
284°  (Kossel).  It  can  be  heated  to  250°  without  melting 
or  suffering  any  decomposition,  but  when  heated  more 
strongly  it  gives  off  white  vapors  which  possess  a  distinct 
iso-nitril  odor,  at  the  same  time  it  carbonizes  and  takes  fire. 
When  evaporated  with  concentrated  nitric  acid,  as  in  the 
ordinary  xanthine  test,  it  gives  only  a  slight  yellow  residue. 
On  the  other  hand,  Weidel's  test,  evaporation  with  chlo- 
rine water  containing  a  trace  of  nitric  acid,  and  then  placing 
the  dry  residue  into  an  ammoniacal  atmosphere  under  a 
bell-jar,  gives  a  beautiful  rose-red  color. 

It  is  difficultly  soluble  in  cold  water  (though  more  easily 
than  xanthine) ;  somewhat  more  readily  soluble  in  hot 
water,  and  insoluble  in  ether  and  alcohol.  It  is  soluble  in 
ammonium  hydrate,  hydrochloric  acid,  and  nitric  acid.  Its 
solutions  are  neutral  in  reaction. 

Silver  nitrate  produces  in  nitric  acid,  as  well  as  in  ammo- 
niacal solutions,  a  flocculent  or  gelatinous  precipitate,  which 
in  concentrated  solutions  forms  an  almost  perfect  jelly-like 
mass.     This  silver  precipitate  is   soluble  in  warm  nitric 


CHEMISTRY    OF    THE    LEUCOMAINES.  325 

acid,  from  which  on  cooling  it  separates  in  white  crystalline 
tufts  possessing  a  silky  lustre.  On  decomposition  with 
hydrogen  sulphide  the  silver  salt  yields  pure  paraxanthine. 
Picric  acid  produces  in  the  hydrochloric  acid  solution  a 
precipitate  consisting  of  densely  felted  yellow  crystalline 
spangles. 

It  is  also  precipitated  by  phosphotungstic  acid  and  copper 
acetate ;  mercuric  chloride  when  added  in  excess  gives  a 
precipitate  composed  of  a  mass  of  colorless  prisms,  which 
are  rather  difficultly  soluble  in  cold  water  ;  easily  in  hot 
water.  The  crystals  of  paraxanthine  mercuric  chloride 
when  moderately  heated  become  opaque  from  loss  of  water 
of  crystallization ;  at  a  higher  temperature  they  melt,  under- 
going at  the  same  time  partial  decomposition,  and  on  strong 
heating  they  evolve  disagreeable  nauseating  vapors.  The 
aqueous  solution  of  this  mercuric  double  salt  gives  with 
silver  nitrate  an  abundant  precipitate  of  silver  chloride, 
which  disappears  on  the  addition  of  ammonium  hydrate 
and  is  replaced  by  the  flocculent  gelatinous  precipitate  of 
silver  paraxanthine.  The  hydrochloric  acid  solution  of 
paraxanthine  crystallizes  with  difficulty  even  when  strongly 
concentrated,  and  on  the  addition  of  platinum  chloride  it 
yields  a  well-crystallizable  orange-colored  paraxanthine 
platinochloride.  It  is  not  precipitated  by  basic  lead  acetate 
nor  by  mercuric  nitrate. 

In  its  behavior  to  the  xanthine  test  this  base  resembles 
hypoxantliine,  whereas  in  giving  Weidel's  reaction  it 
approaches  xanthine.  Finally,  it  coincides  with  guanine 
by  yielding  a  precipitate  with  picric  acid.  Although  it 
thus  agrees  in  some  of  its  reactions  with  all  three  of  these 
xanthine  bodies,  it  can,  however,  be  easily  distinguished 
from  them  by  its  behavior  with  the  fixed  alkalies.  Sodium 
or  potassium  hydrate  dissolves  these  bases  and  holds  them 
in  solution,  but  when  added  to  concentrated  paraxanthine 
solution  the  alkali  produces  a  precipitate  of  long,  glittering, 
crystalline  spangles,  which  under  the  microscope  are  seen  to 
consist  of  delicate  rectangular,  often  longitudinally  striated, 
plates  which  are  either  isolated  or  united  in  tufts.  Besides 
these  crystals  there  are  also  present  hexagonal  plates  resem- 

15 


326  BACTERIAL    POISONS. 

bling  cystin.  Tlie  crystals  are  soluble  in  a  little  water,  or 
on  warming,  but  precipitate  again  on  cooling.  Paraxan- 
thine,  however,  shares  with  heteroxanthine  the  property  of 
forming  a  difficultly  soluble  compound  with  the  fixed  alka- 
lies, but  can  be  distinguished  from  the  latter  by  neutralizing 
with  an  acid  the  solution  of  the  sodium  or  potassium  com- 
pound, when,  in  the  case  of  paraxanthine,  there  will  be 
obtained  a  precipitate  of  the  characteristic  crystals  of  that 
base ;  whereas  heteroxanthine  is  obtained  on  similar  treat- 
ment as  a  dense  pulverulent  precipitate.  This  reaction  is 
not  given  by  theophylline. 

It  is  interesting  to  observe  that  paraxanthine  is  isomeric 
with  theobromine,  theophylline,  and  also  with  a  body  re- 
cently described  by  Fischer  as  dioxy-dimethyl-purpurine. 
In  its  composition  it  is,  therefore,  a  dimethyl-xanthine. 

The  physiological  action  of  paraxanthine  has  been  studied 
by  Salomon.  Injections  into  the  muscles  of  1-2  mg.  pro- 
duced almost  at  once  a  rigor-mortis-like  condition  of  the 
muscles  affected,  with  diminished  reflex  excitability  without 
previous  increase ;  6-8  mg.  introduced  into  the  lymph  sac 
brings  on  a  gradual  loss  of  voluntary  motion  as  well  as  of 
reflex  excitability ;  the  rigor  is  more  marked  in  the  anterior 
extremities,  which  have  a  wooden  or  waxy  consistency. 
Dyspnoea  is  likewise  an  early  symptom,  but  as  soon  as  rigor 
sets  in  the  respirations  drop  far  below  the  normal,  and  may 
even  be  absent  for  several  minutes.  At  times  the  lungs  are 
enormously  dilated,  same  as  in  theobromine.  The  heart's 
action  is  intact  till  the  very  last.  In  mice  the  reflexes  are 
increased  almost  to  a  tetanus.  The  lethal  dose  for  frogs, 
subcutaneously,  was  found  to  be  0.15-0.2  per  cent,  of  the 
body-weight — somewhat  lower  than  that  of  theobromine  and 
xanthine.  The  action  of  these  three  bases  is  very  similar. 
They  produce  in  common  the  slow  creeping  movements, 
followed  by  cessation  of  spontaneous  muscle  action,  com- 
plete loss  of  reflex  excitability  without  a  previous  rise, 
and  the  heart's  action  is  not  affected  till  in  the  latest  stages. 

Carnine,  C7H8N403,  was  isolated  in  1871  from  Amer- 
ican meat-extract  by  Weidel,  but  has  not  been  obtained 


CHEMISTRY    OF    THE    LEUCOMAIJSTES .  327 

from  muscle-tissue  itself.  It  has  also  been  obtained  from 
yeast  liquors  by  Schutzenberger,  and  from  urine  by 
Pouchet.  It  can  be  separated  from  the  meat-extract,  of 
which  it  forms  about  one  per  cent.,  by  the  following  method 
originally  employed  by  Weidel.  The  extract  is  dissolved 
in  six  or  seven  parts  of  warm  water,  then  concentrated 
baryta  water  is  added,  avoiding,  however,  an  excess.  The 
filtrate  is  precipitated  by  basic  lead  acetate.  The  precipitate 
is  collected,  thoroughly  washed  and  pressed,  and  finally  it 
is  repeatedly  extracted  with  a  large  quantity  of  boiling 
water.  The  carnine  lead  salt  is  thus  dissolved  out;  the 
filtrate,  after  removal  of  the  lead  by  hydrogen  sulphide,  is 
evaporated  to  a  small  volume.  The  concentrated  solution 
thus  obtained  is  treated  with  silver  nitrate,  which  gives  a 
precipitate  of  silver  chloride  and  of  the  silver  salt  of  car- 
nine.  By  treatment  with  ammonium  hydrate  the  silver 
chloride  can  be  completely  removed  from  the  precipitate, 
whereas  the  silver  compound  of  carnine  is  insoluble  in  that 
reagent.  To  obtain  pure  carnine  the  silver  salt  is  decom- 
posed with  hydrogen  sulphide,  and  the  filtrate,  after  purifi- 
cation by  bone-black,  is  evaporated  to  crystallization. 

Carnine  forms  white  crystalline  masses,  which  on  drying 
become  loose  and  chalk-like.  It  is  very  difficultly  soluble 
in  cold  water,  easily  and  completely  in  boiling  water,  and 
recrystallizes  on  cooling.  It  is  insoluble  in  alcohol  and 
ether.  The  taste  is  decidedly  bitter,  and  the  reaction  is 
neutral.  The  base  is  not  precipitated  by  neutral  lead 
acetate,  but  is  precipitated  by  the  basic  salt  as  a  flocculent 
white  precipitate,  soluble  in  boiling  water.  On  heating, 
carnine  decomposes  and  takes  fire,  and  at  the  same  time 
gives  off  a  peculiar  odor.  It  crystallizes  with  one  molecule 
of  water,  which  it  loses  at  100°-110°. 

The  hydrochloride,  C7H8N403.HC1,  is  crystalline,  and 
decomposes  on  heating  with  concentrated  hydrochloric  acid. 

The  platinochloride,  C7H8N4O3.HCl.Pt014,  forms  a  fine, 
sandy,  golden-yellow  powder. 

With  silver  nitrate,  carnine  unites  to  form  a  white  floccu- 
lent precipitate,  insoluble  in  nitric  acid  or  in  ammonium  hy- 
drate. Itstbrmula  corresponds  to  2(C7H7  AgN403)  + AgN03. 


328  BACTERIAL    POISONS. 

Carnine  is  not  affected  by  prolonged  boiling  with  concen- 
trated barium  hydrate.  Bromine  water  decomposes  it  with 
the  evolution  of  gas  and  the  formation  of  hypoxanthine. 
This  change  takes  place  according  to  the  following  equa- 
tion : 

C7H8N403  +  2Br  =  C5H4N4O.HBr  +  CH3Br  +  C02. 

A  similar  decomposition  into  hypoxanthine  is  brought  about 
by  the  action  of  nitric  acid,  though  in  this  case  oxalic  acid 
and  a  yellow  body  are'  formed.  When  carnine  is  evaporated 
with  ciilorine  water  containing  a  little  nitric  acid,  the  resi- 
due, on  contact  with  ammonia,  gives  a  rose-red  color 
(murexide  test).  This  is  due,  according  to  Weidel,  to 
the  formation  of  hypoxanthine,  but  it  has  since  been  shown 
that  the  latter  base  does  not  give  this  reaction,  and  hence  it 
is  due  to  the  production  of  xanthine,  or  some  similar  body. 
The  physiological  action  of  carnine  has  been  examined 
somewhat  by  Brucke,  and  according  to  him  it  is  not  very 
poisonous.  The  only  effect  observed,  when  taken  inter- 
nally, was  a  fluctuation  in  the  rate  of  the  heart -beat,  though 
even  this  was  by  no  means  definite  in  its  nature. 

A  Base,  C4H5N50,  was  obtained  by  Gautier  from 
fresh  muscle  tissue  of  beef,  according  to  the  method  given 
on  page  334,  and  on  account  of  a  resemblance  in  some  of 
its  properties  with  xanthine,  he  named  it  pseudoxanthine. 
This  name  is  very  inappropriate,  not  only  because  it  differs 
so  much  in  its  empirical  formula  from  that  of  xanthine, 
(J5H4N402,  but  also  because  the  term  pseudoxanthine  has 
already  been  applied  by  Schuetzen  and  Filehne  to  a 
body  isomeric  with  xanthine,  which  was  obtained  by  the 
action  of  sulphuric  acid  on  uric  acid. 

The  free  base  forms  a  light-yellow  powder,  slightly 
soluble  in  cold  water,  soluble  in  weak  alkali  and  in  hydro- 
chloric acid.  The  hydrochloride  is  very  soluble,  and  it 
forms  stellate  prisms  with  curved  faces,  which  resemble 
the  corresponding  salt  of  hypoxanthine,  and  to  some  extent, 
also,  the  whetstone-shaped  crystals  of  uric  acid. 

Like  xanthine,  its  aqueous  solution  is  precipitated  in  the 


CHEMISTRY    OF    THE    LEUCOMAINES.  329 

cold  by  mercuric  chloride,  silver  nitrate,  and  by  ammo- 
niacal  lead  acetate,  but  not  by  normal  lead  acetate.  On 
evaporation  with  nitric  acid,  the  residue  gives,  on  contact 
with  potassium  hydrate,  as  in  the  case  of  xanthine,  a  beau- 
tiful orange-red  coloration  (xanthine  reaction).  It  differs 
from  xanthine,  not  only  in  its  empirical  composition,  but 
also  in  its  greater  solubility,  and  in  its  crystalline  form. 
It  is  possible  that  this  base,  on  account  of  its  great  resem- 
blance to  xanthine,  may  have  been  mistaken,  at  different 
times,  for  that  compound. 

Gerontijste,  CpH^^,  is  a  new  base  which  was  isolated 
by  Grandis  in  1890.  It  has  been  repeatedly  observed  in 
the  form  of  peculiar  crystals  found  in  the  cell  nuclei  in  the 
liver,  particularly  of  old  dogs.  The  free  base  is  an  isomer 
of  cadaverine,  etc.,  and  resembles  it  somewhat.  It  crystal- 
lizes in  needles  which  are  readily  soluble  in  water  and  alco- 
hol ;  possesses  a  strongly  alkaline  reaction,  and  yields  the 
ordinary  alkaloidal  reactions. 

The  hydrochloride  forms  prismatic  crystals,  which  are 
deliquescent  and  easily  soluble  in  alcohol. 

The  platinochloride,  C5H14N2.2HCl.PtCl4,  is  soluble  in 
water  and  crystallizes  in  spindle-shaped  needles,  arranged 
in  rosettes.     It  decomposes  at  115°. 

The  gold  salt  forms  small  needles,  and  is  easily  soluble 
in  water  and  alcohol. 

It  combines  with  one  molecule  of  mercuric  chloride  to 
form  deliquescent  cubes  or  rectangular  prisms  containing 
two  molecules  of  water  of  crystallization.  It  decomposes 
above  100°.  This  distinguishes  it  from  cadaverine,  which 
combines  with  three  to  four  molecules  of  mercuric  chloride. 
The  crystals  observed  in  the  liver  are  probably  the  phos- 
phate. 

The  new  base  also  yields  a  benzoyl  compound  which 
melts  at  175°-176°. 

Physiological  Action. — It  seems  to  exert  a  paralyzing 
action  upon  the  nerve  centres,  and  leaves  the  nerves  and 
muscles  unaffected. 


330  BACTERIAL    POISONS. 

Spermine,  C2H5N,  or  CinH26N4  (?),  is  the  basic  substance 
obtained  by  Schreiner  (1878)  from  semen,  calf's  heart, 
calf's  liver,  bull's  testicles,  from  the  organs  of  leucocythse- 
mics,  and  also  from  the  surface  of  anatomical  specimens 
kept  under  alcohol.  In  1888  Kunz  reported  the  presence 
of  a  non-poisonous  base,  C2H5N,  spermine  or  ethyleneimide 
in  cholera  cultures.  In  this  case  it  occurs,  then,  as  a  pto- 
maine. A  confirmation  of  the  identity  of  the  two  bases  is 
necessary.  Previous  to  this,  however,  it  had  been  known 
for  a  long  time  under  the  name  of  "  Charcot-Neumann 
or  Leydejst  crystals,"  which  are  the  phosphate  of  spermine. 
These  peculiarly  shaped  crystals  have  been  found  in  the 
sputa  of  a  case  of  emphysema  with  catarrh,  in  the  bronchial 
discharges  in  acute  bronchitis,  as  well  as  in  sputa  of  chronic 
bronchitis,  in  the  blood,  spleen,  etc.',  of  leucocythsemics  and 
ansemics,  and  in  the  normal  marrow  of  human  bones,  as 
well  as  in  human  semen.  Altogether  it  seems  to  have  a 
very  wide  distribution,  especially  in  certain  diseases,  as  in 
leucocythsemia. 

It  can  be  prepared  from  fresh  human  semen  in  the  fol- 
lowing manner :  The  semen  is  washed  out  of  linen  by  a 
little  warm  water,  evaporated  to  dryness,  boiled  with  alco- 
hol, and  the  insoluble  portion  is  allowed  to  subside  by 
standing  some  hours.  The  precipitate  is  filtered  off,  washed, 
and  dried  at  100°.  This  residue,  containing  the  spermine 
phosphate,  is  triturated,  and  then  extracted  with  warm 
ammoniacal  water.  From  this  solution,  on  slow  evapora- 
tion, the  phosphate  crystallizes  in  its  peculiar-shaped 
crystals. 

The  free  base  is  obtained,  on  decomposing  the  phosphate 
with  baryta  and  evaporating  the  filtrate,  as  a  colorless 
liquid,  which,  on  cooling,  crystallizes.  From  alcohol  it 
crystallizes  in  wavellite- shaped  crystals,  which  readily 
absorb  water  and  carbonic  acid  from  the  atmosphere. 
They  are  readily  soluble  in  water  and  in  absolute  alcohol, 
almost  insoluble  in  ether,  and  possess  a  strongly  alkaline 
reaction.  "When  heated  with  platinum  it  gives  off  thick, 
white  fumes,  and  a  weak  ammoniacal  odor.  With  potas- 
sium bismuth  iodide  it  yields    orange-colored  crystalline 


CHEMISTRY    OF    THE    LEUCOM  AINES .  331 

floccules,  which,  under  the  microscope,  appear  as  long, 
sharp,  plumose  needles — distinction  from  diethylenediamine. 
The  aqueous  solution  of  the  base  is  precipitated  by  phos- 
phomolybdic  and  phosphotungstic  acids,  tannic  acid,  gold 
and  platinum  chlorides.  It  cannot  be  volatilized  from 
alkaline  solution  by  steam  without  undergoing  decomposi- 
tion (Majert  and  Schmidt).     It  is  not  poisonous. 

The  hydrochloride,  C2H5]N\HC1  (?),  crystallizes  in  six- 
sided  prisms,  united  in  tufts,  and  is  extremely  soluble  in 
water,  almost  insoluble  in  absolute  alcohol  and  ether. 

The  aurochloride,  C2H5N.HC1. AuCl3  (?),  forms  shining, 
golden-yellow,  irregular  plates,  and  when  freshly  precipi- 
tated it  is  easily  soluble  in  water,  alcohol,  and  ether,  but 
the  dried  salt  is  incompletely  soluble  in  water.  The  aque- 
ous solution,  treated  with  magnesium,  gives  off  a  sperm- 
like odor.     The  platinochloride  crystallizes  in  prisms. 

The  phosphate,  (C2H5N)2.H3P04+3H20(?),  forms  prisms 
and  slender  double  pyramids  arranged  in  rosettes.  It  is 
difficultly  soluble  in  hot  water,  insoluble  in  alcohol,  easily 
soluble  in  dilute  acids,  alkalies,  and  alkali  carbonates.  It 
melts  with  decomposition  at  about  170°.  It  is  probable 
that  the  above  formula  does  not  represent  the  salt  as  found, 
and  from  theoretical  considerations  Ladenburg  was  in- 
clined to  think  that  Schreiner's  phosphate  had  the  com- 
position (C2H5NH)4Ca(P04)2. 

Ladenburg  and  Abel  prepared  in  1888  a  compound, 
ethyleneimine,  which  was  first  supposed  to  be  isomeric  with 
spermine.  The  reaction  whereby  it  is  prepared  is  similar 
to  the  one  by  which  Ladenbtjrg  effected  the  synthesis  of 
piperidine.  Ethylenediamine  hydrochloride  is  subjected  to 
dry  distillation,  when  it  decomposes  into  ammonium 
chloride  and  the  hydrochloride  of  the  new  base.  The  re- 
action was  supposed  to  be  represented  by  the  equation  : 

CH2NH2.HC1    CH2V 
|  =|     >NH.HC1+NH4C1. 

CH2NH2.HC1    CH2 

Since  then  Ladenburg  has  shown  that  the  boiling-point 
of  this  compound  did  not  agree  with  what  it   should  be 


332  BACTEEIAL    POISONS. 

theoretically,  if  represented  by  the  above  formula.  A  de- 
termination of  the  vapor  density  showed  that  the  molecular 
weight  was  twice  that  corresponding  to  the  formula  given, 
and  hence  was  C4H]0]Sr2.  Majert  and  Schmidt  assuming 
spermine  to  be  ethyleneimine,  as  was  apparently  shown  by 
Ladenbueg  and  Abel's  investigation,  attempted  to  pre- 
pare the  latter  on  a  manufacturing  scale  with  the  expecta- 
tion that  it  might  be  used  as  a  substitute  for  Beown- 
Sequabd's  testicular  fluid.  They  were  soon  able  to  show, 
however,  that  ethyleneimine  did  not  possess  the  composition 
assigned  to  it,  but  that  it  was  identical  with  Hofmann's 
diethylenediamine  (piperazine), 

xr/CH2.  CH2\  vTTi 

i^\CH2:CH2/i>'±1- 

This  was  soon  confirmed  by  Hoemann  and  by  Ladenbueg. 
Spermine  was  then  assumed  to  be  identical  with  piperazine, 
but  recently  (1891)  Majert  and  Schmidt  compared  some 
spermine  from  Scheeiner  with  their  own  piperazine  and 
found  the  two  bases  to  be  distinct,  especially  with  reference 
to  the  phosphates  and  the  potassium  bismuth  iodide  pre- 
cipitates. 

About  the  same  time  (1891)  Poehl  announced  that  the 
composition  of  spermine  was  more  complex  than  what  it 
had  been  hitherto  supposed  to  be.  He  ascribed  to  it  the 
formula  C10H26N4.  The  formula  of  the  platinum  salt  cor- 
responded to  C10H26N4.4HC1.2PtCl4 ;  and  that  of  the  gold 
salt  was  represented  by  C10H26N4.4HC1.4AuCl3. 

From  this  it  would  appear  that  spermine  is  essentially 
distinct  from  piperazine.  The  composition  and  structure 
of  this  interesting  base  must  therefore  be  considered  as  not 
settled. 

The  nuclein  of  the  spawn  of  salmon  has  been  found 
by  Miescher  to  exist  in  a  salt-like  combination  with  a 
basic  substance,  to  which  he  applied  the  name  protamine. 
Picard  has  found  it  in  the  same  source,  together  with 
hypoxanthine  and  guanine,  but  no  xanthine.  The  formula 
assigned  to  this  base  is  quite  complex,  and  cannot  be  con- 
sidered  as   definitely   settled.      Analysis   of  the   platino- 


CHEMISTRY    OF    THE    LEUCOMAINES.  333 

chloride  gave :  Pt=24.64,  01=26.45,  N=15.03,  C=22.80, 
H=4.15,  0=6.93.  The  hydrochloride  forms  an  amor- 
phous, hygroscopic,  sticky  mass. 

Leitcomaines  of  the  Creatinine  Group. 

The  knowledge  of  the  formation  of  basic  substances 
(ptomaines)  during  the  putrefaction  of  nitrogenous  organic 
matter,  led  to  a  series  of  investigations  having  for  their 
object  the  isolation  of  alkaloidal  bodies,  if  such  existed, 
from  the  normal  living  tissues  of  the  organism.  A  number 
of  compounds  possessing  alkaloidal  properties,  such  as  the 
xanthine  derivatives,  already  described,  had  been  known 
for  a  long  time,  although  their  physiological  relation  to 
the  animal  economy  was  little,  if  at  all,  understood. 
Guareschi  and  Mosso,  in  the  course  of  their  researches  on 
ptomaines,  were  among  the  first  to  direct  their  attention  to 
the  possible  presence  of  ptomaine-like  bodies  in  fresh  tissues. 
They  obtained  in  those  cases  where  the  extraction  was 
carried  on  without  the  use  of  acids,  only  very  minute  traces 
of  an  alkaloidal  body  (possibly  choline),  and  an  inert  sub- 
stance, methyl-hydantoin,  which,  although  it  can  scarcely 
be  classed  as  a  basic  compound,  is  closely  related  to  creatine, 
and  for  this  reason  will  be  described  at  the  end  of  this  sec- 
tion. Other  Italian  chemists,  as  Paterno  and  Spica  and 
Marino-Zuco,  had  also  shown  that  the  normal  fluids  and 
tissues  of  the  body  were  capable  of  yielding  substances 
alkaloidal  in  nature,  and  these  were  regarded  by  them  as 
identical  with,  or  similar  to,  the  ptomaines  of  Selmi. 

Arginine,  CgH^N/)^  is  a  base  obtained  by  Schulze 
from  the  conglutin  of  lupine  sprouts,  and  according  to  him 
it  is  related  to  creatinine  and  possibly  to  the  leucoma'ines  of 
Gautier.  Lysatine,  C6HI3!N~302,  and  lysatinine,  C6HuN30, 
are  analogous  bases,  obtained  by  Drechsel  from  casein 
(page  242).  These  three  bases  can  properly  be  looked  upon 
as  important  sources  of  the  nitrogenous  bases  found  in 
animals  aud  plants. 

Liebreich,  in  1869,  discovered  in  normal  urine  an 
oxidation-product    of    choline,    probably    identical    with 

15* 


334  BACTEEIAL    POISONS. 

betaine  (pp.  249  and  343),  and  Pouchet,  in  1880;  announced 
the  presence  in  the  same  secretion  of  allantoin,  carnine  (page 
344),  and  an  alkaloidal  base,  which,  however,  was  not 
obtained  at  that  time  in  sufficient  quantity  to  permit  a 
determination  of  its  character.  Subsequently  he  succeeded 
in  isolating  this  base  as  well  as  another  closely  related 
body,  both  of  which  will  be  described  in  their  proper 
place.  Gautier  has  been  engaged  for  a  number  of  years 
in  the  study  of  the  leucomaines  occurring  in  fresh  muscle 
tissue,  and  he  has  succeeded  in  isolating  several  new 
compounds. 

A  number  of  these  substances  are  credited  with  possess- 
ing an  intensely  poisonous  action,  and  if  such  is  the  case 
it  is  very  evident  that  any  undue  accumulation  of  such 
bases  in  the  system,  resulting  from  an  interference  in  the 
elimination,  may  give  rise  to  serious  disturbances.  The 
amount  of  these  substances  present  in  the  daily  yield  of 
the  urine  is  very  small — so  small,  indeed,  that  we  must 
rather  look  upon  this  small  quantity  as  having  escaped 
oxidation  in  the  body.  It  is  well  known  that  the  living 
tissues  possess  an  enormous  oxidizing  and  reducing  power, 
and,  according  to  Gautier,  there  is  constantly  going  on 
in  the  normal  tissues  of  the  body  a  cycle  of  changes — the 
formation  of  leucomaines  and  their  subsequent  destruction 
by  oxidation,  before  they  have  accumulated  in  sufficient 
quantity  to  produce  poisonous  effects. 

The  following  method  was  employed  by  Gautier  in 
his  study  of  the  leucomames  of  muscle  tissue :  The 
finely  divided  fresh  beef-meat  or  the  Liebig's  meat  extract 
is  treated  with  twice  its  weight  of  water,  containing  0.25 
gramme  of  oxalic  acid,  and  one  to  two  c.c.  of  commercial 
peroxide  of  hydrogen  per  litre.  The  purpose  of  these 
precautions  is  to  prevent  fermentation.  At  the  end  of 
twenty-four  hours  the  liquid  is  raised  to  the  boiling-point, 
then  filtered  through  linen,  and  the  residue  is  thoroughly 
squeezed.  The  filtrate  is  again  raised  to  the  boiling-point 
in  order  to  coagulate  any  remaining  albumin,  and  finally 
filtered  through  paper.  The  clear  liquid  thus  obtained  is 
evaporated  in  a  vacuum  at   a  temperature  not  exceeding 


CHEMISTRY    OF    THE    LEUCOM  AINES .  335 

50°,  and  the  acid  syrupy  residue  is  extracted  with  99 
per  cent,  alcohol ;  the  alcoholic  extract  is  in  turn 
evaporated  in  a  vacuum,  and  the  residue  taken  up  with 
warm  alcohol  of  the  same  strength.  The  filtered  alcoholic 
solution  is  set  aside  for  twenty-four  hours,  and  any  deposit 
which  forms  is  removed  by  filtration ;  ether  (65°)  is  then 
added  as  long  as  a  precipitate  continues  to  form,  and  the 
whole  is  again  allowed  to  stand  for  twenty-four  hours. 
The  ether-alcoholic  filtrate  from  this  precipitate  is  evapo- 
rated first  on  the  water  bath,  and  finally  in  a  vacuum ; 
the  slight  residue  obtained  contains  a  small  quantity  of 
basic  substances  possessing  an  odor  of  hawthorn. 

The  syrupy  precipitate  produced  by  the  ether  partially 
crystallizes  on  standing ;  a  little  absolute  ether  is  then 
added,  and  after  standing  several  days  more  the  liquid  is 
separated  by  means  of  an  aspirator  from  the  deposit  of 
crystals  (A).  These  are  first  washed  with  99  per  cent, 
alcohol,  and  then  extracted  with  boiling  95  per  cent, 
alcohol.  The  alcoholic  solution,  concentrated  by  evapora- 
tion, gives,  on  cooling,  a  deposit  of  lemon-yellow-colored 
crystals  of  xantho-creatinine  (B),  from  the  mother-liquor 
of  which  there  separates  a  crop  of  new  crystals  (C).  The 
residue  of  the  crystals  (A)  left  after  treatment  with  the 
boiling  95  per  cent,  alcohol  is  extracted  with  boiling  water, 
which  afterward  gives  a  slight  deposit  of  yellowish- white 
crystals  of  amphi-creatine  (D).  The  aqueous  mother-liquors 
on  concentration  yield  another  deposit  of  orange-colored 
crystals  of  cruso-creatinine  (E).  Gautier  has,  further- 
more, separated  three  other  bases  from  the  mother-liquors 
of  the  crystals  obtained  as  above.  Thus,  a  base  which  he 
named  pseudoxanthine  is  stated  to  have  been  obtained  by 
evaporating  the  alcoholic  mother-liquors  of  B,  D,  E  (?)  in 
a  vacuum,  taking  up  the  residue  with  water,  and  precipi- 
tating the  hot  solution  with  copper  acetate.  The  precipitate 
is  decomposed  with  hydrogen  sulphide,  and  the  aqueous 
solution,  filtered  while  boiling-hot,  yields  a  deposit  of  a 
sulphur-yellow  powder  of  pseudoxanthine.  Thus,  by  the 
use  of  alcohol,  ether,  and  water,  Gautier,  according  to  his 
statement,  has  succeeded  in  obtaining  a  sharp  separation 


336  BACTEKIAL    POISONS. 

between  these  bases.  The  importance  of  the  subject  is  such 
as  to  require  not  only  confirmation  of  the  results  arrived  at 
by  Gautier,  but  also  a  more  detailed  and  exact  study  of 
the  chemical  and  physiological  behavior  of  these  bodies. 

To  the  physiological  chemist  these  substances  are  of 
especial  interest  because  of  the  possible  relation  which  they 
bear  to  the  formation  of  creatine  and  creatinine  in  the 
muscle.  It  will  be  seen  that  in  the  leucomaines  of  this 
group,  as  well  as  in  those  of  the  uric  acid  group,  hydro- 
cyanic acid  plays  a  very  important  part  in  the  molecular 
structure  of  these  bases.  Just  what  the  function  of  this 
cyanogen  group  is,  so  far  as  the  vital  activity  of  the  tissues 
is  concerned,  we  know  very  little,  though  recent  investiga- 
tions seem  to  show  that  the  seat  of  the  cyanogen  formation 
lies  within  the  nucleated  cell,  and  is  intimately  connected 
with  the  functions  of  the  nuclein  molecule. 

Cruso-creatinine,  C5H8N40,  forms  orange-yellow  crys- 
tals which  are  slightly  alkaline  in  reaction,  and  possess  a 
somewhat  bitter  taste.  It  yields  a  soluble,  non- deliquescent 
hydrochloride  crystallizing  in  bundles  of  needles ;  also  a 
soluble  platinochloride  which  forms  tufts  of  beautiful, 
slender  prisms.  The  aurochloride  is  obtained  as  slightly 
soluble,  crystalline  grains,  and,  like  the  platinum  double 
salt,  is  partially  decomposed  on  heating.  It  is  not  precipi- 
tated by  acetate  of  zinc  or  by  mercuric  nitrate,  but  is  pre- 
cipitated in  the  cold  by  solutions  of  alum.  Zinc  chloride 
produces  in  somewhat  concentrated  solutions  a  pulverulent 
precipitate  which  dissolves  on  heating,  and  recrystallizes 
again  when  it  cools.  Like  xantho-creatinine,  it  is  not  thrown 
out  of  solution  by  oxalic  or  nitric  acid,  and  is  thus  distin- 
guished from  urea  and  guanidine  ;  nor  is  it  precipitated  by 
acetate  of  copper — a  distinction  from  xanthine  derivatives. 
Mercuric  chloride  produces  an  abundant  flocculent  precipi- 
tate which  on  heating  partially  dissolves,  decomposing  at 
the  same  time.  Sodium  phosphomolybdate  gives  a  heavy 
yellow  precipitate,  whereas  potassium  mercuro-chloride  and 
iodine  in  potassium  iodide  have  no  eifect.  Potassium  ferri- 
cyanide  is  not  reduced.     This  base  differs  in  its  composition 


CHEMISTRY    OF    THE     LEUCOM AINES .  337 

from  creatinine  by  HCN,  the  elements  of  hydrocyanic  acid, 
but  in  its  crystalline  form  and  alkaline  reaction,  and  some 
other  properties,  it  would  seem  to  be  closely  related  to  this 
latter  substance.  Because  of  this  apparent  relationship  and 
its  golden-yellow  color,  Gautier  named  it  cruso-creatinine. 

Xantho-creatinuste,  C5H10N4O,  is  the  most  abundant 
of  muscle  leucoma'ines.  It  crystallizes  in  sulphur-yellow, 
thin  spangles,  consisting  of  nearly  rectangular  plates  which 
resemble  somewhat  those  of  cholesterin.  It  is  soft  and 
talc-like  to  the  touch  ;  possesses  a  slightly  bitter  taste,  and 
when  dissolved  in  boiling  alcohol  it  gives  off  the  odor  of 
acetamide,  though  ordinarily  in  the  cold  it  has  a  slight 
cadaveric  odor.  When  heated,  the  substance  evolves  an 
odor  of  roast  meat,  carbonizes  in  part,  and  yields  ammonia 
and  methylamine.  The  crystals  are  amphoteric  in  reaction, 
are  soluble  in  cold  water,  and  can  be  recrystallized  from 
boiling  99  per  cent,  alcohol. 

It  forms  a  hydrochloride  crystallizing  in  plumose  needles, 
and  a  very  soluble  platinochloride ;  the  aurochloride  crys- 
tallizes with  difficulty.  Like  creatinine,  it  is  precipitated 
by  zinc  chloride ;  the  yellowish-white  precipitate  dissolves 
with  partial  dissociation  on  warming,  and  on  cooling  sepa- 
rates as  isolated  or  stellate  groups  of  fine  needles  which 
possess  the  composition  (C5H10N4O)2ZnC]3.  Silver  nitrate 
throws  down,  in  the  cold,  a  flocculent  precipitate  which 
likewise  dissolves  on  heating,  and  recrystallizes  in  needles. 
Mercuric  chloride  produces  a  yellowish-white  precipitate. 
It  is  not  precipitated  by  oxalic  or  nitric  acid,  nor  by  potas- 
tassio-mercuric  chloride,  or  iodine  in  potassium  iodide. 
Tannin  produces  in  time  a  slight  turbidity,  while  sodium 
phosphomolybdate  gives  a  heavy  yellowish  precipitate. 
This  base  is  distinguished  from  the  members  of  the  uric 
acid  group  by  not  giving  a  precipitate  with  copper  acetate, 
not  even  on  heating. 

On  gentle  oxidation  with  potassium  permanganate  it  is 
converted  into  a  black  substance  insoluble  in  acids  and 
alkalies,  and  resembling  azulmic  acid.  By  treatment  with 
recently  precipitated  mercuric  oxide,  it  yields  a  substance 


338  BACTERIAL    POISONS. 

which  can  be  recrystallized  from  boiling  93  per  cent, 
alcohol  in  needles  which  possess  a  slight  alkaline  reaction, 
and  forms  a  slightly  soluble,  crystalline  platinochloride. 
This  new  substance  is  precipitated  from  alcoholic  solution 
by  the  addition  of  ether,  as  a  mass  of  beautiful,  white,  silky 
needles  resembling  caffeine.  These  crystals  melt  at  174°  ; 
caffeine  melts  at  178°. 

Xantho-creatine,  given  in  fairly  large  doses,  is  poison- 
ous, producing  in  animals  depression,  somnolence,  and 
extreme  fatigue,  accompanied  by  frequent  defecation  and 
vomiting.  In  its  general  properties  this  base  resembles 
creatinine  very  much,  and  it  was  on  account  of  this  resem- 
blance and  its  yellow  color  that  it  was  named  xantho-crea- 
tinine.  This  relation  becomes  especially  evident  since  this 
base  appears  in  the  physiologically  active  muscle  at  the 
same  time  with  creatinine,  constituting  sometimes  one-tenth 
of  the  creatinine  present.  Monari  has  found  this  base  in 
the  aqueous  extract  of  the  muscles  of  an  exhausted  dog, 
and  also  in  the  urine  of  soldiers  tired  by  several  hours' 
march.  He  also  demonstrated  its  presence  in  the  urine  of  a 
dog  after  previous  injection  of  creatinine. 

Amphi-creatine,  C9H19N704,  is  slightly  soluble  and 
crystallizes  from  boiling  water  in  yellowish-white  oblique 
prisms,  which  possess,  if  any,  a  slightly  bitter  taste. 
When  heated  to  100°  it  decrepitates  somewhat,  and  at 
110°  it  becomes  opaque  white.  Potassium  hydrate  does 
not  decompose  it  in  the  cold.  Although  a  weak  base,  it 
combines  to  form  salts  just  as  the  preceding  members  of 
this  group.  The  hydrochloride  is  crystalline,  and  is  not 
deliquescent ;  the  platinochloride  forms  rhombic  plates, 
which  are  soluble  in  water,  but  are  insoluble  in  absolute 
alcohol ;  the  aurochloride  crystallizes  in  easily  soluble,  very 
small,  microscopic  crystals,  which  are  tetrahedral  to  hexa- 
hedral  in  their  habit.  It  is  not  precipitated  by  copper 
acetate  or  by  mercuric  chloride ;  nor  does  it  give  the 
murexide  test,  or  the  xanthine  reaction.  Sodium  phospho- 
molybdate  produces  a  yellow,  pulverulent  precipitate.  In 
its  properties  it  resembles  creatine,  and  indeed  Gautier 


CHEMISTRY    OF    THE    LEUCOIAINES.  339 

thinks  it  may  be  possibly  a  combination  of  creatine, 
C4H9N302,  and  a  base  C5H]0N4O2,  which,  it  will  be  seen, 
differs  from  the  former  only  by  a  HCIST  group.  This 
second  compound,  if  it  really  exists,  has  an  analogy  in 
cruso-creatinine,  the  relation  of  which  to  creatinine  may  be 
expressed  by  the  equation  : 

C6H8K"40  =  C4H7N30+HCN. 

Cruso-creatinine.  Creatinine. 

In  a  similar  manner,  amphi-creatine  may  be  regarded  as 
C9H19N704  =  2C4H9¥302+HCN. 

Amphi-creatine.  Creatine. 

A  Base,  ChH^N^O^  was  isolated  by  Gatjtier  from 
the  mother- liquors  of  xantho-creatinine.  It  crystallizes  in 
colorless  or  yellowish,  thin,  apparently  rectangular  plates, 
which  are  tasteless,  and  possess  an  amphoteric  reaction. 
The  hydrochloride  forms  bundles  of  fine  needles;  the  sul-' 
phate  yields  a  confused  mass  of  needles ;  the  platinochlo- 
ride  is  soluble,  non-deliquescent,  and  crystalline.  When 
heated  with  water  in  a  sealed  tube  at  180°-200°,  it  gives 
off  ammonia  and  carbonic  acid,  and  is  converted  into  a 
new  base,  which,  however,  has  not  been  studied.  This 
reaction  may  be  expressed  by  the  equation  : 

C„HMN10OB  =  2C5H10N4O2+CO(NH2), 

Urea. 

The  urea  which  at  first  forms,  is,  in  turn,  decomposed, 
thus  : 

CO(NH2)2+H20  =  C02+2NH3. 

It  is  to  be  observed  that  this  base  differs  iu  composition 
from  the  following  one  by  HON,  the  hydrocyanic  acid 
molecule. 

A  Base,  0]2II25NnO5,  was  obtained  from  the  mother- 
liquors  of  cruso-creatinine,  and  forms  rectangular  silky 
plates,  resembling  those  of  the  preceding  base  and  of 
xantho-creatinine.     It  forms  crystallizable  salts. 

These  complex  bases  will  require  further  study  in  order 


340  BACTERIAL    POISONS. 

to  elucidate  their  physiology,  and  the  possible  connection 
which  they  may  have  with  the  formation  of  urea,  and  of 
the  creatinine  derivatives  already  described. 

Methyl-hydantoin,  C4H6N202,  =  CO/^1^'^, 

— This  substance  was  obtained  by  Guareschi  and  Mosso 
(1883),  by  extracting  fresh  meat  with  1—1.5  volumes  of 
water  (without  addition  of  acid),  for  two  hours  at  50°-60°. 
The  aqueous  extract  was  evaporated  on  the  water-bath  and 
the  residue  was  extracted  with  95  per  cent,  alcohol.  This 
alcoholic  solution,  after  the  alcohol  was  driven  off,  was 
taken  up  in  water,  filtered,  and  the  aqueous  solution  was 
first  extracted  with  ether,  then  rendered  alkaline  with 
ammonia,  and  again  extracted  with  ether.  The  alkaline 
ether  extract  gave  on  evaporation  a  white  crystalline  residue 
of  methyl-hydanto'in.  The  amount  of  this  substance 
present  in  flesh  appears  to  be  quite  variable,  since,  at  times, 
none  whatever  can  be  extracted.  Albertoni  has  isolated 
it  from  dog's  flesh.  Previous  to  its  discovery  in  flesh  by 
Guareschi  and  Mosso,  it  was  known  for  a  long  time  as  a 
decomposition-product  of  various  nitrogenous  bases  of  the 
body.  Thus,  Neubauer  prepared  it  by  heating  creatin- 
ine with  barium  hydrate,  while  Huppert  obtained  it  by 
fusing  together  sarcosine  with  urea.  As  it  occurs  in  muscle 
it  is  probably  derived  from  the  creatine,  though  under 
what  conditions  this  splitting  up  takes  place  is  not  definitely 
known.  Acetic  and  lactic  acids  are  incapable  of  effecting 
this  change.  At  all  events,  it  belongs  to  the  ureides,  and 
is  intermediate  between  creatinine,  sarcosine,  and  urea 
Compare  the  above  formula  with  that  of  creatinine,  p.  226. 
Methyl-hydanto'in  forms  prisms  which  are  easily  soluble 
in  water  and  alcohol,  and  but  slightly  soluble  in  cold  ether. 
It  melts  at  156°  (Salkowski)  ;  at  159°-160°  (Guareschi 
and  Mosso).  Its  aqueous  solution  is  slightly  acid  in  reac- 
tion. On  strong  heating  it  volatilizes.  When  fused  with 
potassium  hydrate  it  gives  off  ammonia;  it  reduces  mercuric 
nitrate  in  the  cold.  Treated  with  mercuric  oxide  it  assumes 
an  alkaline    reaction,  and  the    filtrate    on    heating  yields 


CHEMISTRY    OF    THE    LEUCOMAINES.         341 

metallic  mercury.  With  silver  oxide  it  forms  pearly  lanceo- 
late plates  having  the  composition  C4H5N202.Ag.  It  does 
not  give  any  alkaloidal  reactions. 

Undetermined  Leucomaines. 

Leucomaines  of  Expired  Air. 

It  was  shown  at  quite  an  early  period  that  exhalations 
from  animals  contain,  besides  an  increased  amount  of  car- 
bonic acid,  some  organic  matter,  the  nature  of  which,  on 
account  of  the  exceedingly  minute  quantity  in  which  it 
occurs,  has  never  been  satisfactorily  determined.  Never- 
theless, various  observers  did  not  hesitate  to  ascribe  to  it 
the  ill  effects  consequent  upon  breathing  impure  air,  while 
at  the  same  time  the  carbonic  acid  formed  during  respira- 
tion was  considered  as  either  entirely  inert  or  as  insignifi- 
cant in  its  action.  Thus,  respired  air  from  which  moisture 
and  carbonic  acid  have  been  removed,  but  which  still  contains 
the  organic  vapors,  has  been  found  to  be  highly  poisonous. 
On  the  other  hand,  if  the  respired  air  is  drawn  through 
a  red-hot  tube  to  destroy  the  organic  matter,  the  air  thus 
purified  is  capable  of  sustaining  life  even  in  presence  of  a 
large  percentage  of  carbonic  acid.  While  it  cannot  be, 
therefore,  doubted  that  the  organic  matter  of  expired  air 
plays  a  most  important  part  in  producing  the  well-known 
noxious  effects  resulting  from  breathing  confined  and  vitiated 
air,  nevertheless  it  would  seem  from  experiments  made  by 
Angus  Smith  that  the  increase  of  even  such  small  quanti- 
ties of  carbonic  acid  in  the  air,  as  from  0.04,  the  normal 
amount  present,  to  0.1  per  cent.,  is  capable  of  producing 
systemic  disturbances  characterized  by  a  decrease  in  the 
pulse-rate  and  an  increase  in  the  rate  of  respiration. 

Smith  is  consequently  of  the  opinion  that  the  constant 
lowering  of  the  pulse  in  impure  air  occasioned  by  the  pres- 
ence of  carbonic  acid  must  have  a  depressing  effect  on 
the  vitality.  Whatever  ill  effects  the  carbonic  acid  may 
produce  of  itself,  it  remains  certain  that  this  gas  is  not  the 
most  potent  and  most  injurious  constituent  of  respired  air ; 


342  BACTERIAL    POISONS. 

and  the  investigations  of  Hammond,  Nowak,  Seegen, 
and  others,  point  conclusively  to  the  organic  matter  as  the 
direct  and  immediate  agent  which  produces  those  symp- 
toms of  sickness  and  nausea  experienced  in  badly  ventilated 
closed  rooms. 

Of  special  importance  to  the  sanitarian  and  physician  is 
the  work  on  the  nature  and  action  of  the  poisonous  principle 
of  expired  air  which  has  been  done  by  Brown-Sequard, 
d'Arsonval,  and  R.  Wurtz.  The  first  two  observers 
found  that  the  vapors  exhaled  by  dogs,  when  condensed, 
and  the  aqueous  liquid  (20-44  c.  c.)  thus  obtained  was  in- 
jected into  other  animals,  death  was  produced,  generally 
within  twenty- four  hours.  The  symptoms  observed  were 
dilatation  of  the  pupil,  increase  of  heart- beat  to  240-280 
per  minute,  which  may  last  for  several  days  or  even  weeks, 
while  the  temperature  remains  normal ;  the  respiratory 
movements  are  generally  slowed,  and  usually  there  is  ob- 
served paralysis  of  the  posterior  members.  Choleraic  diar- 
rhoea is  invariably  present.  As  a  rule,  it  appears  that 
larger  doses  cause  labored  respiration,  violent  retching,  and 
contraction  of  the  pupil.  A  rapid  lowering  of  temperature, 
0.5°  to  5°,  is  sometimes  observed.  These  same  symptoms, 
apparently  in  aggravated  form,  were  obtained  when  the 
liquid  had  been  previously  boiled  for  the  purpose  of  de- 
stroying any  germs  that  might  be  present.  The  appearances 
presented  on  post-mortem  were  much  like  those  observable 
in  cardiac  syncope. 

The  above  work  has  been  confirmed,  in  part,  by  R. 
Wurtz,  who,  by  passing  expired  air  through  a  solution  of 
oxalic  acid,  has  obtained  besides  ammonia  a  volatile  organic 
base  which  is  precipitated  by  Bouchardat's  reagent  and 
by  potassio-mercuric  iodide.  It  is  said  to  form  a  platinum 
double  salt  crystallizing  in  short  needles,  and  a  soluble 
gold  salt.  When  heated  to  100°  it  gives  off  a  peculiar 
odor.  This  basic  substance  may  properly  be  looked  upon 
as  a  leucoma'ine. 

Dastre  and  Loye  and  Lehmann  and  Jessen  have 
repeated  the  above  experiments  with  wholly  negative  re- 
sults.    It  is  possible  that  the  most  highly  poisonous  sub- 


CHEMISTRY    OF    THE    LEUCOM AINES.         343 

stances  formed  in  the  body  when  there  is  an  insufficient 
air- supply  are  not  eliminated  in  the  exhaled  air. 

Sewer-air,  according  to  observations  made  by  Odling, 
contains  a  basic  substance  which  is  probably  in  composition 
a. compound  ammonia.  It  contains,  however,  more  carbon 
than  methylamine  and  less  than  ethylamine. 

It  should  be  remarked  that  Jackson  has  (Dec.  1887) 
announced  the  presence  in  expired  air  of  quantities  of  car- 
bon monoxide  gas  sufficient  to  produce  the  ill  effects  ordi- 
narily attributed  to  the  organic  matter.  The  presence  of 
this  poisonous  gas  must  first  be  fully  demonstrated  before 
it  can  be  taken  into  account  in  the  consideration  of  the 
toxicity  of  air ;  certainly,  even  if  present,  it  cannot  explain 
the  results  obtained  by  the  French  investigators  as  stated 
above. 

According  to  Ilosva,  expired  air  contains  nitrous  acid. 
This  may  possibly  be  derived  from  that  which  is  constantly 
being  formed  in  the  mouth,  probably  by  the  reduction  of 
nitrates  (Miller). 

Leucomaines  of  the  Urine. 

A  number  of  basic  substances  have  been  isolated  at 
different  times  from  the  urine,  and  on  that  account  they 
may  be  properly  classed  as  leucomaines.  Thus,  Liebreich 
(1869)  found  in  the  urine  a  base  which  apparently  was  an 
oxidation-product  of  choline,  and  which  has  since  been 
regarded  as  identical  with  betaine.  In  1866  Dupre  and 
Bence  Jones  found,  among  other  things  in  the  urine,  an 
alkaloidal  body  which  in  sulphuric  acid  solution  possessed 
a  blue  fluorescence  (see  p.  347).  Most  of  the  members  of 
the  uric  acid  group  of  leucomaines  have  been  detected  in 
the  urine  and  on  account  of  their  well-defined  nature  they 
are  described  by  themselves.  In  the  urine  and  feces  of 
cystinuria  Udranszky  and  Baumann  discovered  the  well- 
known  ptomaines,  cadaverine  and  putrescine.  For  isola- 
tion, see  pp.  207  and  208. 

In  1879,  Thudichum  announced  the  presence  in  the 
urine  of  four  new  alkaloids,  one  of  which,  urotheobromine, 


344  BACTERIAL    POISONS. 

was  subsequently  rediscovered  by  Salomon  and  named 
paraxanthine  (page  321).  Another  base  which  was  ob- 
tained, namely,  reducine,  yielded  a  barium  salt  which  readily 
reduced  the  salts  of  silver  and  mercury.  Its  formula  prob- 
ably corresponds  to  C12H24N609  or  C6HuN"304.  A  third 
alkaloid,  parareducine,  formed  a  zinc  compound  having  the 
composition  C6H9N3O.ZnO.  A  fourth  base  is  said  to  give 
a  compound  with  platinum  chloride  and  to  contain  an  aro- 
matic nucleus  (aromine).  Besides  these  four  bases  Thudi- 
chum  describes  two  other  substances  which  he  considers 
basic.  These  are  urochrome,  the  normal  pigment  of  the 
urine,  and  creatinine. 

In  1880,  Pouchet  announced  the  presence  of  carnine, 
C7H8N403,  and  of  another  base  which  he  subsequently  ana- 
lyzed and  found  to  have  either  the  composition  C7H12N402 
or  C7H14N402.  This  substance  formed  deliquescent  fusi- 
form crystals,  sometimes  crystallized  in  bundles  or  irregular 
spheres,  which  possessed  a  slightly  alkaline  reaction  and 
combined  with  acids  to  form  crystallizable  salts.  It  was 
soluble  in  dilute  alcohol,  almost  insoluble  in  strong  alcohol, 
insoluble  in  ether.  The  hydrochloride  yielded  double  salts 
with  gold  chloride,  platinum  chloride,  and  mercuric  chlo- 
ride. The  platinochloride  formed  deliquescent  golden- 
yellow  rhombic  prisms.  This  base  occurred  in  the  dialysate 
(see  page  265).  From  the  non-dialyzable  portion,  Pouchet 
obtained  another  base  corresponding  to  the  formula 
C3H5N02,  which  he  calls  the  "extractive  matter  of  urine." 
It  yields  precipitates  with  the  general  alkaloidal  reagents, 
is  non-crystallizable  and  is  altered  on  exposure  to  air  and 
resinified  by  hydrochloric  acid.  On  the  addition  of  plati- 
num chloride  it  is  rapidly  oxidized,  but  does  not  yield  a 
platinochloride.  The  same  author  regards  the  urine  as 
containing  very  small  quantities  of  some  pyridine  bases 
which  are  analogous  or  identical  with  those  obtained  by 
Gautier.  and  Etard  from  decomposing  fish. 

The  distinguished  Italian  toxicologist  Selmi  was,  per- 
haps, the  first  to  draw  attention  to  the  probable  formation 
of  basic  substances  in  the  living  body  during  those  patho- 
logical changes  brought  on  by  the  presence  of  pathogenic 


CHEMISTRY    OF    THE    LEUCOM  AINES .  345 

germs ;  and  in  a  memoir  presented  to  the  Academy  of 
Sciences  of  Bologna,  in  December,  1880,  he  announced 
that  infectious  diseases,  or  those  in  which  there  occurs  an 
internal  disarrangement  of  some  element,  either  plasmic  or 
histological,  must  be  accompanied  or  followed  by  an  elimi- 
nation of  more  or  less  characteristic  products,  which  would 
be  a  sign  of  the  pathological  condition  of  the  patient.  To 
support  this  theory  he  examined  a  number  of  pathological 
urines,  and  succeeded  in  obtaining  from  them  basic  sub- 
stances, some  of  which  were  poisonous,  others  not.  Thus, 
a  specimen  of  urine  from  a  patient  with  progressive  paraly- 
sis gave  two  bases  strongly  resembling  nicotine  and  coniine ; 
from  other  pathological  urines  the  bases  obtained  usually 
had  either  an  ammoniacal  or  trimethylamine  odor.  A 
strong  confirmation  of  Selmi's  theory  is  seen  in  the  obser- 
vations made  by  Bouchard,  Villiers,  Lepine,  Gau- 
tier,  and  others,  all  of  whom  have  found  basic  substances 
in  the  urine  of  various  diseases. 

It  is  now  a  well-established  fact  that  the  urine  of  disease, 
as  cholera  (Bouchard)  and  septicaemia  (Feltz),  etc.,  is  far 
more  poisonous  than  normal  urine.  That  poisons  which 
are  generated  within  the  body  by  the  activity  of  bacteria 
can  be  excreted  in  the  urine  is  seen  in  the  fact  that  im- 
munity to  the  action  of  bacillus  pyocyaneus  has  been  con- 
ferred on  animals  by  previous  injection  of  urine  taken  from 
animals  inoculated  with  that  bacillus  (Bouchard)  or  with 
filtered  cultures  of  the  same  (Charrix  and  Buffer). 

Unfortunately,  none  of  these  bases  supposedly  character- 
istic of  pathological  urines  have  been  isolated  in  a  chemi- 
cally pure  condition ;  nor  has  the  study  of  normal  urine 
been  carried  sufficiently  far  to  show  the  positive  absence  of 
such  bodies. 

Villiers  has  denied  the  existence  of  alkaloids  in  normal 
urine,  and  this  has  been  confirmed  experimentally  by 
Stadthagejst,  who,  moreover,  agreed  with  Feltz  and 
Ritter  that  specific  organic  poisons  are  absent  from  nor- 
mal urine.  The  observed  physiological  action  is  there- 
fore largely  (70-80  per  cent.),  or  wholly,  due  to  the  potas- 
sium salts  present. 


346  BACTERIAL    POISONS. 

Leucomaines  of  the  Saliva. 

According  to  the  statement  of  Gautier  (1881),  normal 
human  saliva  contains  divers  toxic  substances  in  small 
quantities  which  differ  very  much  in  their  action  according 
to  the  time  of  their  secretion,  and  probably  according  to 
the  individual  gland  in  which  they  are  secreted.  The 
aqueous  extract  of  saliva  at  100°  is  poisonous  or  narcotic 
in  its  action  toward  birds.  To  show  the  presence  of  basic 
substances,  the  aqueous  extract  was  slightly  acidulated  with 
dilute  hydrochloric  acid,  then  precipitated  by  Mayer's 
reagent;  the  precipitate  was  washed,  then  decomposed  by 
hydrogen  sulphide,  and  the  solution  filtered.  The  filtrate 
on  evaporation  gave  a  residue  consisting  of  microscopic 
slender  needles  of  a  soluble  hydrochloride.  This  salt, 
purified  by  extraction  with  absolute  alcohol,  forms  soluble 
crystalline,  but  easily  decomposable  double  salts  with 
platinum  chloride  and  with  gold  chloride.  The  solution 
of  the  hydrochloride  produces  an  immediate  precipitate  of 
Prussian  blue  in  a  mixture  of  potassium  ferricyanide  and 
ferric  chloride,  and  when  injected  into  birds  produces  a 
condition  of  stupor. 

Leucomaines  from  other  Tissues  of  the  Body. 

Selmi's  work  upon  the  formation  of  ptomaines  during 
the  process  of  putrefaction  led  many  investigators  to  doubt 
the  production  of  these  bases  by  the  decomposition  of  the 
proteid  or  other  complex  molecules.  To  substantiate  this, 
a  number  of  chemists,  especially  Italian,  endeavored  to 
show  that  Selmi's  bases,  to  a  large  extent  at  least,  exist 
preformed  in  the  various  tissues.  Paterno  and  Spica 
(1882)  succeeded  in  extracting  from  fresh  blood  as  well  as 
from  fresh  albumin  of  eggs  substances  identical,  or  at  least 
similar,  to  those  designated  under  the  name  of  ptomaines. 
Their  observations,  however,  were  confined  to  the  detection 
of  alkaloidal  reactions  in  the  various  extracts  obtained  by 
Dragendorff's  method,  and  at  no  time  were  they  in 
possession   of  a   definite   chemical  individual.     Marino- 


CHEMISTRY    OF    THE    LEUCOMAINES.         347 

Zuco  (1885)  was  more  successful,  inasmuch  as  he  succeeded 
in  obtaining  from  fresh  tissues  and  organs  relevant  quan- 
tities of  a  base  identical  with  choline,  and,  in  addition,  he 
obtained  extremely  minute  traces  of  other  alkaloidal  bodies. 
One  of  these,  obtained  by  the  Stas  method  from  the  liver 
and  spleen  of  an  ox,  exhibited  in  hydrochloric  acid  solution 
a  beautiful  violet  fluorescence  resembling  very  much  that 
of  the  salts  of  quinine.  A  similar  base,  probably  identical 
with  this  one,  was  obtained  by  Bence  Jones  and  Dupre 
(1856)  from  liver,  nerves,  tissues,  and  other  organs,  and 
was  named  by  them  "animal  chinoidine."  A  greenish- 
blue  fluorescence  is  frequently  observable  in  the  alcoholic 
extracts  of  decomposing  glue  as  well  as  from  other  putrefy- 
ing substances.  From  a  number  of  very  thorough  experi- 
ments, he  concluded  that  basic  substances  do  not  preexist 
in  fresh  organs,  but  that  the  acids  employed  in  the  process 
of  extraction  exert  a  decomposing  action  upon  the  lecithin 
present  in  the  tissues,  resulting  in  the  formation  of  choline. 
He  further  showed  that  the  method  of  Dragendorff,  on 
account  of  the  larger  quantity  of  extractives  which  form, 
invariably  gave  a  larger  yield  of  this  base  than  did  the 
Stas-Otto  method.  Similar  observations  were  made  by 
Guareschi  and  Mosso,  by  Coppola  and  others.  At  the 
present  time  there  is  no  doubt  that  some  basic  substances, 
among  these  choline,  can  be  formed  by  the  action  of  re- 
agents, and,  on  the  other  hand,  it  is  equally  well  demon- 
strated that  similar  bases  do  preexist  in  the  physiological 
condition  of  the  tissues  and  fluids  of  the  body. 

Recently  R.  Wurtz  has  obtained  from  normal  blood  a 
number  of  crystalline  products  of  alkaline  reaction,  which 
form  well-crystallizable  double  salts  with  gold,  platinum, 
and  mercuric  chlorides.  These,  however,  have  not  been 
as  yet  subjected  to  analysis,  because  of  the  minute  quan- 
tities which  were  isolated. 

Morelle  (1886)  showed  the  presence  in  the  spleen  of 
the  ox  of  a  base,  the  hydrochloride  of  which  crystallized 
in  deliquescent  needles  and  likewise  formed  crystalline 
platino-  and  aurochlorides.  From  experiments  made  by 
Laborde,  the  base  would  seem  to  possess  decided  toxic 


348  BACTERIAL    POISONS. 

properties,  bringing  on  a  dyspnoeic  condition  with  con- 
vulsive movements  and  loss  of  motion.  The  post-mortem 
examinations  revealed  an  extended  visceral  oedematous 
infiltration,  and  stoppage  of  the  heart  in  systole. 

A.  W.  Blyth  has  claimed  to  have  isolated  from  milk 
two  alkaloidal  substances,  namely  galactine,  the  lead  salt 
of  which  is  said  to  have  the  formula  Pb203C54H18N4025, 
and  lactochrome,  the  mercury  salt  of  which  is  represented 
by  the  formula  HgOC6H18N06. 

Leucomdines  of  the  Venoms  of  Poisonous  Serpents. 

The  study  of  the  chemistry  of  the  venoms  of  serpents 
and  of  batrachians  is  fraught  with  so  many  difficulties  and 
with  so  much  danger,  that  we  cannot  wonder  at  the  present 
unsatisfactory  condition  of  our  knowledge  in  regard  to  the 
poisonous  principles  which  they  contain.  Much  of  the  work 
that  has  been  done  hitherto  is  not  only  inaccurate  and  very 
contradictory,  but  is  far  from  meeting  the  requirements  of 
exact  toxicological  research.  From  recent  investigations 
it  seems,  however,  to  be  quite  certain  that  the  most  active 
constituent  of  the  venom  of  serpents  is  not  alkaloidal  in 
its  nature  as  has  been  supposed  by  some.  In  1881 
Gautier  announced  the  isolation  of  two  alkaloids  from 
the  venom  of  the  cobra  which  gave  precipitates  with  tannin, 
Mayer's  reagent,  Nessler's  reagent,  iodine  in  potas- 
sium iodide,  etc.  They  formed  crystallizable  platinochlo- 
rides  and  aurochlorides,  and  also  crystalline,  neutral,  some- 
what deliquescent  hydrochlorides.  The  neutral  or  slightly 
acid  solutions  produced  an  immediate  precipitate  of  Prus- 
sian blue  in  a  mixture  of  potassium  ferricyanide  and  ferric 
chloride.  These  substances  possess  a  decided  physiological 
action,  though  Gautier  himself  does  not  consider  them 
to  be  the  most  dangerous  constituents  of  the  venoms.  This 
observation  of  Gautier  as  to  the  presence  of  distinct  basic 
substances  in  venoms  is  at  variance  with  that  of  Wolcott 
Gibbs,  who  has  been  unable  to  obtain  an  alkaloid  from  the 
rattlesnake  (Crotalus)  venom.  S.  Weir  Mitchell  and 
E.  T.  Reichert  likewise  state  that  they  have  been  utterly 


CHEMTSTEY    OF    THE    LEUCOM  AINES.         349 

unable  to  substantiate  Gautier's  statements.  Still  more 
recently  Wolfenden,  in  an  elaborate  paper  on  the  nature 
of  cobra  venom,  has  confirmed  Wolcott  Gibbs  as  to  the 
entire  absence  of  any  alkaloidal  body. 

Mitchell  aud  Reichert  have  made  a  careful  study  of 
the  venoms  of  various  serpents,  such  as  cobra,  rattlesnake, 
moccasin,  and  Indian  viper,  and  have  succeeded  in  isolating 
two  proteid  constituents,  one  belonging  to  the  class  of 
globulins  and  the  other  to  the  peptones.  The  peptone  is 
said  to  be  non-precipitable  by  alcohol.  According  to 
them,  the  globulin  constituent  consists  of  at  least  three 
distinct  globulins.  They  found  that  boiling  coagulates 
and  destroys  the  globulin  as  a  poison,  but  leaves  the 
venom  peptone  toxically  unchanged,  so  that  the  solution, 
though  still  poisonous,  fails  to  produce  the  characteristic 
local  lesions  due  to  fresh  or  unboiled  venom.  On  the  other 
hand,  Gautier  asserts  that  the  venom  is  not  sensibly 
altered  on  being  heated  to  120°-125°,  and  that  the  toxic 
action  remains  constant  even  when  all  the  proteid  con- 
stituents are  removed,  thus  showing  that  the  toxic  action 
cannot  be  attributed  to  the  albuminoids.  The  venom  pep- 
tone from  the  rattlesnake  or  the  moccasin,  however,  when 
injected  into  animals  produced  toxic  effects  which  were 
marked  by  an  oedematous  swelling  over  the  site  of  injection ; 
the  tumor  was  filled  with  serum,  and  so  also  was  the  sub- 
cutaneous cellular  tissue.  Furthermore,  a  gradual  breaking 
down  of  the  tissues  occurred,  accompanied  by  rapid  putre- 
factive changes  and  a  more  or  less  extensive  slouch.  That 
peptones  may  possess  intensely  poisonous  properties  has  been 
shown  to  be  the  case  by  a  number  of  authors,  among  whom 
may  be  mentioned  ScHMiDT-MtJLHEor,  Hofmeister, 
Pollitzer,  and  others.  Brteger  has,  moreover,  demon- 
strated that  the  formation  of  peptones  in  the  process  of 
digestion  is  accompanied  by  the  development  of  a  toxic 
ptomaine  which  he  has  named  psptotoxine. 

The  venom  globulins,  on  the  other  hand,  though  present 
in  less  quantity  than  the  peptones,  induced  the  same  re- 
markable local  effects  seen  on  injection  of  the  pure  venom. 

16 


350  BACTERIAL    POISONS. 

They  cause  local  bleedings,  destroy  the  coagulability  of  the 
blood,  and  rapidly  corrode  the  capillaries. 

These  results  of  Mitchell  and  Reichert,  which  are 
given  here  somewhat  in  full,  have  been  questioned  by 
WoLFENDEisr,  who,  while  agreeing  in  the  main  that  the 
poisonous  property  of  venom  is  due  to  proteid  constituents, 
regards  their  peptone  not  as  a  true  peptone,  but  rather  as 
one  or  more  bodies  of  the  albumose  group  of  proteids.  He 
likewise  regards  the  globulin  of  moccasin  venom  to  be 
some  other  proteid  body.  According  to  him,  the  cobra 
venom  owes  its  toxicity  to  the  proteids,  globulin,  serum- 
albumin,  acid  albumin.  Occasionally  there  seem  to  be 
present  traces  of  peptone  and  of  hemialbumose. 

Brieger  was  at  first  apparently  inclined  to  believe  that 
the  action  of  venoms  is  due  to  animal  alkaloids,  on  the  ground 
that  these  bases  are  extremely  soluble,  and  hence  always  go 
into  solution  along  with  the  likewise  very  soluble  proteid 
constituents,  and  that  the  difficulty  in  their  isolation  lies  in 
the  elimination  of  these  proteids.  Since  then  Brieger 
and  Frankel  pointed  out  the  poisonous  nature  of  some 
bacterial  proteids,  and  also  showed  that  cobra  poison  yields 
with  alcohol  a  precipitate  which  gives  proteid  reactions. 

The  proteids  of  serpents'  venom  should  be  compared 
with  the  poisonous  proteids  formed  by  the  activity  of  the 
pathogenic  bacteria,  as  well  as  with  similar  compounds, 
the  phytalbumoses  of  castor  seeds,  jequirity,  etc.  Possibly 
similar  compounds  will  be  found  in  croton  and  other 
species  of  ricinus,  jatropha,  loco-weed,  etc.  The  poisons 
secreted  by  certain  spiders  and  fish  may  be  mentioned  in 
this  connection. 

Cloez  and  Gratiolet  in  1852  examined  the  poison 
contained  in  the  cutaneous  pustules  of  some  batrachians, 
and  succeeded  in  extracting  a  substance  which  gave  a  white 
precipitate  with  mercuric  chloride  and  formed  a  platinum 
double  salt.  Beyond  this  meagre  information  very  little  is 
known  in  regard  to  the  character  of  these  poisons,  though 
Zalesky,  in  1866,  annouuced  the  isolation  of  an  alkaloid 
to  which  he  assigned  the  formula  C34H60lS"2O5,  and  which 
he  named  salamandarine.    According  to  Dutartre  (1890) 


CHEMISTRY    OF    THE    LEUCOM  AINES . 


351 


this  base  is  a  leucomaine.  and  similar  products,  but  with 
different  physiological  actiou,  are  to  be  found  in  other 
batrachians,  as  the  toad,  triton(?),  green  and  red  frogs, 
and  in  the  epidermis  of  some  fish.  According  to  Calmeil, 
the  poison  from  the  toad  contains  methyl-carbylarnine  and 
isocyanacetic  acid. 


Table  of  Leucomaines. 


Formula. 

Name. 

Discoverer. 

Source. 

Physiological  action. 

C5  H5  N5 

Adenine. 

Kossel. 

Nuclein-contain- 
ing  organs. 

Non-poisonous;  muscle 
stimulant. 

C5  H4  N4  0 

HypoxanthiDe. 

Scherer. 

Nuclein-contain- 
ing  organs. 

Non-poisonous ;  muscle 
stimulant. 

C6  H6  N6  0 

Guanine. 

Unger. 

Nuclein-contain- 
ing  organs, 
guano. 

Non-poisonous ;  muscle 
stimulant. 

C5  H4  N4  02 

Xanthine. 

Marcet. 

Nuclein-contain- 
ing  organs, 
calculi. 

Non-poisonous ;  muscle 
stimulant. 

C6  H6  N4  02 

Heteroxanthine. 

Salomon. 

Urine. 

C7  H8  N4  02 

Paraxanthine. 

Thudichum 
Salomon. 

Poisonous. 

C7  H8  N4  03 

Carnine. 

Weidel. 

Liebig's  meat 
extract. 

Non-poisonous ;  muscle 
stimulant. 

C4  H6  N6  0 

Pseudoxanthine(?) 

Gautier. 

Muscle. 

C5  H14N2 

Gerontine. 

Grandis. 

Liver  of  dogs. 

Poisonous. 

C2H6N(?) 

Spermine. 

Schreiner. 

Sperma,  in  tis- 
sues of  leuco- 
cythsemics. 

Non-poisonous. 

C6  H8  N4  0 

Cruso-creatiniue. 

Gautier. 

Muscle. 

C5  H10N4  0 

Xantho-creatinine 

" 

" 

Poisonous. 

C9  H19N7  04 

Amphi-creatine. 

" 

" 

C1]H24N10O5 

Unnamed. 

" 

" 

C12Hs5N„05 

" 

" 

" 

C7  H12N4  02 

" 

Pouchet. 

Urine. 

C3  H6  N02 

" 

" 

" 

C34H60N2  05 

Salamandarine. 

Zalesky. 

Salamander. 

Poisonous. 

CHAPTER   XIII. 

THE  AUTOGENOUS   DISEASES. 

All  living  things  are  composed  of  cells.  The  simplest 
forms  of  life  are  unicellular,  and  in  these  all  the  functions 
of  life  devolve  upon  the  single  cell.  Absorption,  secretion, 
and  excretion  must  be  carried  on  by  the  same  cell.  A 
collection  of  unicellular  organisms  might  be  compared  to  a 
community  of  men  with  every  individual  his  own  tailor, 
shoemaker,  carpenter,  cook,  farmer,  gardener,  blacksmith, 
etc.  However,  only  the  lowest  forms  of  life  are  unicellular; 
all  others  are  multicellular.  In  the  higher  animals  there 
is  a  differentiation  not  only  in  the  size  aud  structure  of  the 
cells,  but  in  the  labor  which  they  perform.  The  body  of 
man  may  be  compared  to  a  community  in  which  labor  has 
been  specialized.  Certain  groups  of  cells,  which  we  desig- 
nate by  the  term  "  organ,"  take  upon  themselves  the  task 
of  doing  some  special  line  of  work,  the  well-doing  of  which 
is  essential  to  the  health,  not  only  of  that  group,  but  of 
other  groups  as  well,  or  of  the  body  as  a  whole.  There 
is  an  interdependence  among  the  various  organs.  Certain 
groups  of  cells  supply  the  fluids  or  juices  which  act  as 
digestants,  and  among  these  there  is  again  a  division  of 
labor.  The  salivary  glands  supply  a  fluid  which  partially 
digests  the  starch  of  our  food  ;  the  peptic  glands  supply 
the  gastric  juice  which  does  the  preliminary  work  in  the 
digestion  of  the  proteids ;  while  the  pancreatic  juice  com- 
pletes the  digestion  of  the  starches  begun  in  the  mouth,  of 
the  proteids  begun  in  the  stomach,  and  does  the  special 
work  of  emulsifying  the  fats.  But  even  some  of  these 
products  of  complete  digestion  would  be  harmful  should 
they  enter  the  circulation  unchanged.  The  peptones  must 
be  converted  into  serum-albumin  by  the  absorbing  mechan- 
ism of  the  walls  of  the  intestines,  and  while  10  per  cent. 


THE    AUTOGENOUS    DISEASES.  853 

of  the  fat  of  the  food  is  split  up  into  glycerin  and  fatty 
acids  by  the  action  of  the  pancreatic  juice,  a  much  smaller 
per  cent,  enters  the  thoracic  duct  in  this  divided  form. 
The  food  may  be  taken  in  proper  quality  and  quantity ; 
the  digestive  juices  may  do  their  work  promptly  and 
properly,  but  if  the  absorbents  fail  to  perform  their  func- 
tions properly,  disease  results.  It  may  happen  that  the 
failure  lies  in  improper  or  imperfect  assimilation  and  the 
result  becomes  equally  disastrous,  and  with  the  effects  of 
non-elimination  we  are  fairly  conversant.  Of  the  myriads 
of  cells  in  the  healthy  human  body  there  are  none  which 
are  superfluous.  It  is  true  that  among  these  ultimate 
entities  of  existence,  death  is  constantly  occurring,  but  in 
health  regeneration  goes  on  with  equal  rapidity  and  each 
organ  continues  to  do  its  daily  and  hourly  task.  The 
microscope  has  made  us  familiar  with  the  size  and  shape 
of  the  various  cells  of  the  body,  and  students  of  pathology 
have  described  the  alterations  in  form  and  size  character- 
istic of  various  disease  states.  But  we  must  remember 
that  in  the  study  of  these  ultimate  elements  of  life  there 
are  other  things,  besides  their  morphological  history,  to 
investigate.  They  are  endowed  with  life,  and  they,  as  well 
as  the  germs,  have  a  physiology  and  chemistry  which  we 
know  but  slightly.  They  are  influenced  beneficially  or 
harmfully,  as  the  case  may  be,  by  their  environment. 
They  grow  and  perform  their  functions  properly  when 
supplied  with  the  needed  pabulum.  They  are  not  immune 
to  poisonous  agents.  They  are  injured  when  the  products 
of  their  own  activity  accumulate  about  them. 

The  object  in  writing  this  chapter  has  been  to  collect 
what  evidence  we  may  concerning  those  diseases  which 
arise  from  imperfect  or  improper  activity  of  the  cells  of  the 
body,  not  due  to  the  introduction  of  foreign  cells.  To 
designate  this  class  of  diseases  we  have  selected  the  word 
autogenous,  and  we  understand  that  in  these  diseases  the 
materies  morbi  is  a  product  of  some  cell  of  the  body,  and 
not,  as  in  the  case  of  the  infections  diseases,  of  cells  intro- 
duced from  without  the  body. 

It  is  true,  without  exception  so  far  as  we  know,  that  the 


354  BACTERIAL    POISONS. 

excretions  of  all  living  things,  plants  and  animals,  contain 
substances  which  are  poisonous  to  the  organisms  which 
excrete  them.  A  man  may  drink  only  chemically  pure 
water,  eat  only  that  food  which  is  free  from  all  adultera- 
tions, and  breathe  nothing  but  the  purest  air,  free  from  all 
organic  matter,  both  living  and  dead,  and  yet  that  man's 
excretions  would  contain  poisons.  Where  do  these  poisons 
originate?  They  are  formed  within  the  body.  They 
originate  in  the  metabolic  changes  by  which  the  complex 
organic  molecule  is  split  up  into  simpler  compounds.  We 
may  suppose — indeed,  we  have  good  reasons  for  believing — 
that  the  proteid  molecule  has  certain  lines  of  cleavage  along 
which  it  breaks  when  certain  forces  are  applied,  and  that 
the  resulting  fragments  have  also  lines  of  cleavage  along 
which  they  break  under  certain  influences,  and  so  on  until 
the  end-products,  urea,  ammonia,  water,  and  carbon-dioxide 
are  reached  ;  also  that  some  of  these  intermediate  products 
are  highly  poisonous  has  been  abundantly  demonstrated. 
The  fact  that  the  hydrocyanic  acid  molecule  is  a  frequent 
constituent  of  the  leucoma'ines  is  one  of  great  significance. 
We  know  that  chemical  composition  is  an  indication  of 
physiological  action,  and  the  intensely  poisonous  character 
of  some  of  the  leucoma'ines  conforms  to  this  fact.  It 
matters  not  whether  the  proteid  molecule  be  broken  up  by 
organized  ferments,  bacteria,  or  by  the  unorganized  fer- 
ments of  the  digestive  juices,  by  the  cells  of  the  liver  or  by 
those  still  unknown  agencies,  which  induce  metabolic 
changes  in  all  the  tissues — in  all  cases  poisons  may  be 
formed.  These  poisons  will  differ  in  quality  and  quantity 
according  to  the  proteid  which  is  acted  upon,  and  according 
to  the  force  which  acts. 

Peptones  formed  during  digestion  do  not  in  health  reach 
the  general  circulation.  When  injected  directly  into  the 
blood  they  act  as  powerful  poisons.  They  destroy  the 
coagulability  of  the  blood,  lower  blood-pressure,  and  in 
large  quantities  cause  speedy  death.  Brunton  attributes 
the  lassitude,  depression,  sense  of  weight  in  the  limbs,  and 
dulness  in  the  head  occurring  in  the  well-fed,  inactive  man, 
after  his  meals,  to  poisoning  with  peptones.     The  remedy 


THE    AUTOGENOUS    DISEASES.  355 

which  he  proposes  is  less  food,  especially  less  nitrogenous 
food,  and  more  exercise.  That  some  substance  resulting 
from  the  proteids  of  the  food  is  the  cause  of  this  trouble 
Brunton  thinks  is  evidenced  by  the  fact  that  the  weak- 
ness and  languor  are  apparently  less  after  meals  consisting 
of  farinaceous  foods  only. 

That  peptone  finds  its  way  into  the  general  circulation 
frequently  is  shown  by  its  detection  in  the  urine  in  many 
diseased  conditions,  some  of  which  are  infectious  and  others 
autogenous  in  character.  However,  propeptonuria,  or  albu- 
mosuria, is  more  common  than  peptonuria,  and  we  have 
already  seen  that  many  of  the  bacterial  albumoses  are  among 
the  most  highly  poisonous  bodies  known,  but  the  action  of 
the  albumoses  formed  during  digestion  has  not,  so  far  as 
we  know,  been  studied.  The  valuable  work  of  Kuhne  and 
Chittenden  on  the  chemical  character  of  these  bodies 
should  be  supplemented  by  a  thorough  investigation  of  their 
physiological  effects  when  injected  into  the  blood.  It  is  more 
than  probable  that  valuable  information  would  be  secured 
by  such  studies.  That  albumose  is  frequently  found  in  the 
urine  is  shown  by  the  following  list  of  diseases  in  which 
it  has  been  observed,  given  in  the  last  edition  of  the  work 
of  Neubauer  and  Vogel  on  the  urine  :  Kosner  has 
found  it  in  spermatorrhoea ;  Koppen,  in  mental  diseases 
without  spermatorrhoea  ;  Kahler,  in  osteomalacia;  Bence 
Jones,  in  multiple  myeloma ;  Senator  and  others,  in 
dermatitis,  intestinal  ulcer,  liver  abscess,  croupous  pneu- 
monia, apoplexy,  vitium  cordis,  resectio  coxse,  parame- 
tritis, endocarditis,  typhoid  fever,  nephritis,  phthisis,  etc. ; 
Loeb,  in  measles  and  scarlet  fever ;  Leube,  in  urticaria ; 
and  Lassar,  after  inunctions  of  petroleum.  Kottnitz, 
Furstner,  and  others,  find '  albumose  frequently  in  the 
urine  in  mental  diseases.  Evidently,  there  is  much  to 
learn  from  the  study  of  the  conditions  accompanied  by  the 
elimination  of  the  albumoses  in  the  urine.  It  is  more  than 
probable  that  the  acute  Bright's  disease  following  scarlet 
fever,  diphtheria,  and  the  other  acute  infectious  diseases, 
owes  its  existence  to  the  poisonous  albumoses  of  these  dis- 
eases.    Prior   has  recently    shown    that  undigested  egg 


356  BACTERIAL    POISONS. 

albumin  is  sometimes  absorbed  and  produces  marked  dis- 
turbances. A  boy,  after  eating  sixteen  raw  eggs,  had  a 
high  fever  accompanied  by  the  appearance  of  both  albumin 
and  haemoglobin  in  the  urine. 

Brieger  obtained  by  digesting  fibrin  with  gastric  juice 
a  substance  which  gives  reactions  with  many  of  the  general 
alkaloidal  reagents  and  to  which  he  has  given  the  name 
"  peptotoxine."  A  few  drops  of  a  dilute  aqueous  solution 
of  this  substance  sufficed  to  kill  frogs  within  fifteen  min- 
utes. The  frogs  became  apparently  paralyzed  and  did  not 
respond  to  stimuli.  Slight  tremor  was  perceptible  in  the 
muscles  of  the  extremities.  Rabbits  of  about  one  kilo- 
gramme weight  were  given  from  0.5  to  1  gramme  of  the 
extract  subcutaneously.  About  fifteen  minutes  after  the 
injection,  paralysis  beginning  in  the  posterior  extremities 
set  in ;  the  animal  fell  into  a  somnolent  condition,  sank 
and  died.  In  some  rabbits  several  hours  elapsed  before 
the  above-mentioned  symptoms  appeared. 

Peptotoxine  was  found  by  Brieger  to  be  formed  not 
only  by  the  digestive  juice,  but  to  be  among  the  first 
putrefactive  products  of  proteids,  as  fibrin,  casein,  brain 
substance,  liver,  and  muscle. 

It  is  highly  probable  that  many  of  the  nervous  symptoms 
which  accompany  some  forms  of  dyspepsia  are  due  to  the 
formation  and  absorption  of  poisonous  substances. 

In  some  persons  the  tendency  to  the  formation  of  poisons 
out  of  certain  foods  is  very  marked.  Thus,  there  are  some 
to  whom  the  smallest  bit  of  egg  is  highly  poisonous  ;  with 
others,  milk  will  not  agree ;  and  instances  of  this  kind  are 
sufficiently  numerous  to  give  rise  to  the  adage,  "  What  is 
one  man's  meat  is  another  man's  poison." 

Brunton  is  of  the  opinion  that  the  condition  which  we 
term  "  biliousness,"  and  which  is  most  likely  to  exist  in 
those  who  eat  largely  of  proteids,  is  due  to  the  formation 
of  poisonous  alkaloids ;  but  of  this  we  have  no  positive 
proof. 

Whether  or  not  the  unorganized  digestive  ferments  ever 
find  their  way  into  the  blood  in  quantity  sufficient  to  cause 
deviations  from  health,  we  are  not  in  a  position  to  state 


THE    AUTOGENOUS    DISEASES.  357 

definitely.  The  older  physiological  chemists  teach  us  that 
pepsin  and  trypsin  are  frequent,  if  not  constant  constituents 
of  normal  urine,  but  their  experiments  were  made  without 
any  reference  to  the  possibility  of  the  ferments  which  they 
found  being  formed  by  the  bacteria  of  the  urine,  and  after 
carefully  going  over  the  literature  of  the  subject  we  are  not 
prepared  to  pass  judgment  on  the  truth  of  their  statements. 
However  this  may  be,  the  fact  that  these  ferments  manifest 
a  marked  toxicological  eifect  when  introduced  into  the 
blood  is  of  great  interest,  especially  at  this  time.  Hilde- 
brandt  has  recently  reported  the  results  of  some  experi- 
ments made  by  himself  upon  this  subject.  He  finds  that 
a  fatal  dose  of  pepsin  for  dogs  is  from  0.1  to  0.2  gramme 
per  kilogramme  of  body  weight.  The  subcutaneous  injec- 
tion of  these  quantities  is  followed  by  a  marked  elevation 
of  temperature,  which  he  designates  as  "  ferment  fever." 
This  fever  begins  within  an  hour  after  the  injection, 
reaches  its  maximum  after  from  four  to  six  hours,  and 
may  continue  for  some  days.  On  the  day  preceding  death, 
the  temperature  generally  falls  below  the  normal.  During 
the  period  of  elevation  there  are  frequent  chills. 

The  symptoms  which  accompanying  the  fever  vary 
somewhat  with  the  species  of  animal.  Rabbits  lose  flesh 
nothwithstanding  the  fact  that  they  continue  for  a  while 
to  eat  well,  they  become  very  weak,  and  death  is  preceded 
by  convulsive  movements.  Dogs  tremble  in  the  limbs,  be- 
come uncertain  in  gait,  and  vomiting,  dyspnoea  and  coma 
are  followed  by  death. 

On  section  there  is  observed  parenchymatous  degenera- 
tion of  the  muscles  of  the  heart  and  similar  changes  in  the 
liver  and  kidney.  There  are  abundant  hemorrhages  in  the 
intestinal  canal,  in  Peyeh's  patches,  in  the  mesenteric 
glands ;  and  in  the  lungs  in  cats.  Thrombi  are  frequently 
found  in  the  lungs  and  in  some  cases  in  the  kidneys. 

The  effect  upon  the  coagulability  of  the  blood  is  worthy 
of  note.  At  first  there  is  a  period  during  which  the  coagu- 
lability of  the  blood  is  greatly  lessened,  then  follows  a 
period  of  greater  rapidity  in  coagulating,  and  it  is  in  this 
latter  stage  that  the  thrombi  are  formed. 

16* 


358  BACTEEIAL    POISONS. 

These  experiments  are  interesting  not  only  as  a  possible 
explanation  of  the  cause  of  some  of  the  autogenous  fevers, 
which  will  be  discussed  later,  but  in  view  of  the  present 
tendency  to  inject  such  complex  animal  solutions  as 
Brown-S^quard's  elixir  and  Koch's  lymph  subcutane- 
ously,  and  they  will  probably  cause  us  to  exercise  a  little 
more  care  in  this  direction. 

That  certain  febrile  conditions  are  autogenous  there  can 
be  no  doubt.  These,  like  other  diseases  originating  within 
the  system,  may  be  due  to  either  of  the  following  causes  : 

1.  There  may  be  an  excessive  formation  of  poisonous  sub- 
stances in  the  body.  Thus,  Bouchard  has  shown  that 
the  urine  excreted  during  the  hours  of  activity  is  much 
more  poisonous  than  that  excreted  during  the  hours  of  rest. 
Both  physical  and  mental  labor  are  accompanied  by  the 
formation  of  these  deleterious  bodies,  and  if  the  hours  of 
labor  are  prolonged  and  those  of  rest  shortened,  there  will 
be  an  accumulation  of  effete  matters  within  the  system. 

2.  The  accumulation  of  the  poisonous  matters  may  be  due 
to  deficient  elimination.  3.  Some  organ  whose  duty  it  is 
to  change  harmful  into  harmless  bodies  may  fail  to  prop- 
erly perform  its  functions.  Illustrations  of  diseased  con- 
ditions arising  from  these  several  causes  will  be  given. 

First,  we  may  mention  fatigue  fever,  which  is  by  no 
means  uncommon,  and  from  which  the  overworked  physi- 
cian not  infrequently  suffers.  One  works  night  and  day 
for  some  time;  elimination  seems  to  proceed  normally; 
but  after  a  few  days  there  is  an  elevation  of  temperature 
of  from  one  to  three  degrees,  the  appetite  is  impaired,  and 
then  if  the  opportunity  for  rest  is  at  hand  sound  and  rest- 
ful sleep  is  impossible.  The  tired  man  retires  to  his  bed 
expecting  to  fall  asleep  immediately,  but  he  tosses  from  side 
to  side  all  night,  or  his  sleep  is  fitful  and  unrefreshing. 
The  brain  is  excited  and  refuses  to  be  at  rest.  The  senses 
are  alert,  and  all  efforts  to  sink  them  in  repose  are  unavail- 
ing. Fatigue  fever  is  frequently  observed  in  armies  upon 
forced  marches,  especially  if  the  troops  are  young  and  un- 
accustomed to  service.     Mosso  has  studied  this  fever  in 


THE    AUTOGENOUS    DISEASES.  359 

the  Italian  army.  He  states  that  in  fatigue  the  blood  is 
subjected  to  a  process  of  decomposition  brought  about  by 
the  infiltration  into  it  from  the  tissues  of  poisonous  sub- 
stances, which,  when  injected  into  the  circulation  of  healthy 
animals,  induce  malaise  and  all  the  signs  of  excessive  ex- 
haustion. It  is  possible  that  in  this  decomposition  of  the 
blood  the  fibrin-ferment,  which,  according  to  Schmidt,  is 
held  in  combination  in  the  colorless  corpuscles,  is  liberated ; 
and  it  has  been  shown  by  Edelbeeg  that  the  injection  of 
small  quantities  of  free  fibrin-ferment  into  the  blood  causes 
fever,  while  the  injection  of  larger  quantities  is  followed  by 
the  formation  of  thrombi,  as  has  been  demonstrated  by  the 
experiments  of  Edelberg,  Bonne,  Birk,  and  Kohlar. 

Fatigue  fever  is  often  accompanied,  especially  during  the 
period  of  elevation,  by  chilly  sensations,  and  consequently 
it  is  pronounced  malarial  and  quinine  is  administered,  but 
it  does  no  good — often  harm,  by  increasing  cerebral  excite- 
ment. The  proper  treatment  is  prolonged  rest,  with  proper 
attention  to  elimination. 

Then  there  is  the  fever  of  exhaustion,  which  differs  from 
fatigue  fever  only  in  degree.  It  is  brought  on  by  pro- 
longed exertion  without  sufficient  rest  and  often  without 
sufficient  food.  The  healthy  balance  between  the  formation 
and  elimination  of  effete  matter  is  disturbed,  and  it  may 
be  weeks  before  it  is  reestablished---indeed,  it  may  never 
be  regained,  for  some  of  those  cases  terminate  fatally.  The 
fever  of  exhaustion  may  take  on  the  typhus  form,  delirium 
may  appear,  muscular  control  of  the  bowels  may  be  lost, 
and  death  may  result. 

That  the  fever  of  exhaustion  has  been  mistaken  for 
typhoid  by  some  of  the  ablest  clinical  teachers  is  shown  by 
Peter  in  the  following  quotation.  "It  was  in  1852,"  says 
he,  "  when  entering  upon  my  clinical  studies  and  ardent  in 
my  attendance  at  the  clinic  of  Chomee,  I  was  witness  of 
the  following  instance :  A  young  man  was  received  under 
the  celebrated  professor's  charge  suffering  from  prostration, 
muscular  pain,  and  rhachialgia.  Chomel  made  the  exam- 
ination with  all  the  care  and  attention  used  by  him  ;  then 


360  BACTERIAL    POISONS. 

— as  was  also  usual  with  him  in  the  presence  of  the  patient 
— he  gave  the  diagnosis  in  Latin,  which  was  lAut  febris 
peyeriea,  aut  variola  incipientis'  (either  typhoid  fever  or 
incipient  smallpox).  I  felt  rather  dissatisfied  at  a  diagnosis 
so  little  precise  by  one  so  eminent  in  his  art.  The  truth  of 
the  matter  was,  though  Chomel  was  not  aware  of  it,  this 
young  fellow  in  a  state  of  destitution  had  walked  from 
Compiegne  to  Paris,  sleeping  by  the  wayside  at  night  and 
nourishing  himself  with  such  refuse  food  as  chance  supplied. 
It  was  under  such  circumstances  the  patient  had  developed 
febrile  symptoms.  The  day  after  his  admission,  and  simply 
from  rest  in  bed,  he  felt  better,  and  the  day  following  he 
was  altogether  well." 

That  all  cases  of  the  fever  of  exhaustion  do  not  terminate 
so  rapidly  as  that  instanced  above  many  physicians  know. 
We  have  seen  at  least  one  such  case  terminate  fatally. 

Then,  again,  there  is  the  fever  of  non-elimination,  which 
all  physicians  of  experience  have  observed.  There  is  a 
feeling  of  languor,  the  head  aches,  the  tongue  is  coated,  the 
breath  offensive,  and  the  bowels  constipated.  The  physi- 
cian fears  typhoid  fever,  but  finds  that  a  good,  brisk  cathar- 
tic dissipates  all  unpleasant  symptoms,  and  the  temperature 
falls  to  the  normal.  This  fever  is  also  liable  to  appear 
among  those  who  are  confined  to  bed  from  other  causes. 
Brunton  says  :  "  No  one  who  has  watched  cases  of  acute 
diseases,  such  as  pneumonia,  can  have  failed  to  see  how  a 
rise  of  temperature  sometimes  coincides  with  the  occurrence 
of  constipation,  and  is  removed  by  opening  the  bowels." 
The  surgeon  and  obstetrician  have  often  had  cause  to  rejoice 
when  they  have  found  a  fever,  which  they  feared  indicated 
septicaemia,  disappearing  after  free  purgation. 

Bouchard  has  shown  that  normal  feces  contain  a  highly 
poisonous  substance,  which  may  be  separated  from  them  by 
dialysis,  and  which,  when  administered  to  rabbits,  produces 
violent  convulsions.  He  estimates  that  the  amount  of 
poisonous  alkaloids  formed  in  the  intestines  of  a  healthy 
man  each  twenty-four  hours  would  be  quite  sufficient  to 
kill,  if  it  was  all  absorbed.     He  proposes  the  term  "  ster- 


THE    AUTOGENOUS    DISEASES.  361 

corsenaia"  for  that  condition  which  results  from  arrest  of 
excretion  from  the  intestine. 

It  is  more  than  probable  that  the  poisons  of  the  intes- 
tines are  due  to  the  bacteria  which  are  normally  present ; 
but  this  would  not  exclude  the  fever  of  non- elimination 
from  the  list  of  autogenous  diseases.  The  bacterial  cells 
which  are  normally  present  in  the  intestines  cannot  be 
regarded  as  invaders  from  without. 

It  would  seem  from  some  recent  studies  that  not  all  sur- 
gical fevers  are  due  to  bacterial  activity.  The  absorption 
of  aseptic  blood-clots  and  of  disintegrated  tissue  in  cases  of 
complicated  fractures  and  contusions  of  the  joints  is  accom- 
panied by  an  elevation  of  the  temperature  above  normal. 
A  like  result  may  follow  the  intravenous  injection  of  a 
sterile  solution  of  haemoglobin  or  of  the  blood  of  another 
animal.  The  causative  agent  in  the  production  of  these 
fevers  remains  unknown.  In  the  blood  of  twelve  out  of 
fifteen  patients  with  aseptic  fever,  at  the  clinic  of  Noth- 
nagel,  Hammeeschlag  has  found  free  fibrin -ferment, 
but  in  five  persons  without  fever  he  found  the  same  sub- 
stauce  in  the  blood.  This  leaves  the  causative  agent  in  the 
production  of  the  aseptic,  or,  more  properly  speaking,  the 
non-bacterial,  fevers  unknown. 

The  chemical  theory  of  so-called  uraemia  has  received 
support  in  recent  researches,  notwithstanding  the  fact  that 
the  old  idea  that  urea  is  the  active  poison  and  the  theory 
of  Feeeeiches  that  ammonium  carbonate  is  the  active 
agent  have  been  abandoned. 

Landois  laid  bare  the  surface  of  the  brain  in  dogs 
and  rabbits,  and  sprinkled  the  motor  area  with  creatine, 
creatinine,  and  other  constituents  of  the  urine.  Urea, 
ammonium  carbonate,  sodium  chloride,  and  potassium 
chloride  had  but  slight  effect ;  but  creatine,  creatinine, 
and  acid  sodium  phosphate  caused  clonic  convulsions  on 
the  opposite  side  of  the  body  which  later  became  bilateral. 
The  convulsions  continued  at  intervals  for  from  two  to 
three  days,  when,  growing  gradually  weaker,  they  disap- 
peared.    Landois  concludes  that  chorea  gravidarum  is  a 


362  BACTERIAL    POISON'S. 

forerunner  of  eclampsia.      These  experiments  have  been 
confirmed  by  Leubuscher  and  Zeichen. 

Falck  injected  into  both  sound  and  nephrotomized  ani- 
mals fresh  urine,  urine  and  the  ferment  of  Muscuxus  and 
Lea,  and  urine  which  had  undergone  spontaneous  decom- 
position, without  producing  any  symptoms  which  were 
comparable  with  those  observed  in  uraemia.  However,  he 
did  find  that  if  a  few  drops  of  an  infusion  of  putrid  flesh 
were  added  to  the  urine  before  injection  all  the  typical 
symptoms  of  uraemia  were  induced.  That  the  infusion  of 
putrid  flesh  alone  had  no  effect  was  also  demonstrated. 
This  would  lead  us  to  believe  that  some  ferment  in  the 
infusion  converts  some  constituent  of  the  urine  into  a 
highly  poisonous  body.  In  this  connection  attention  may 
be  called  to  the  fact  that  creatine  may  be  converted  by  the 
action  of  certain  germs  into  methyl  -guauidine,  which  pro- 
duces convulsions.  Whether  such  conversion  occurs  in 
uraemia  or  not,  and  if  it  does  what  the  cause  of  it  is,  are 
questions  which  must  be  left  for  future  investigations  to 
decide.  It  would  be  well  for  someone  to  test  the  brain 
and  blood  of  a  person  who  has  died  in  ursemic  convulsions 
for  methyl-guanidine. 

That  there  is  a  marked  disturbance  of  tissue  metabolism 
caused  by  the  inhalation  of  vitiated  air  has  been  shown  by 
Araki.  In  the  urine  of  animals  rendered  unconscious  by 
being  kept  in  a  confined  space  this  experimenter  found 
albumin,  sugar,  and  lactic  acid.  If  the  animals  had  been 
kept  without  food  for  some  days  before  being  subjected  to 
this  experiment  albumin  and  lactic  acid  were  found,  but 
no  sugar  appeared.  This  was  undoubtedly  due  to  the  fact 
that  the  glycogen  of  the  body  had  been  exhausted  by  the 
fasting.  Identical  results  were  observed  in  animals  which 
were  poisoned  with  carbon  monoxide.  Dogs  which  were 
poisoned  with  curare,  and  in  which  the  respiratory  move- 
ments .were  maintained  artificially,  secreted  very  little 
urine ;  but  the  blood  was  found  to  contain  considerable 
quantities  of  sugar  and  lactic  acid.  The  urine  of  frogs  in 
which  the  respiration  was  retarded  by  the  production  of 
tetanus  with  strychnine  secreted  urine  containing  sugar  and 


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THE    AUTOGENOUS    DISEASES.  363 

lactic  acid.  In  the  urine  of  three  epileptics  there  were 
found  albumin  and  lactic  acid  directly  after  the  seizure. 
The  factor  common  to  all  these  cases  is  diminished  oxygen- 
ation of  the  blood,  and  to  this  is  ascribed  the  appearance  of 
the  abnormal  constituents  of  the  urine.  These  investiga- 
tions demonstrate  the  influence  of  impure  air  upon  the 
chemistry  of  the  living  cells  of  the  animal  body. 


CHAPTER    XIV. 

bibliogeaphy. 
Ptomaines. 

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2d  Ed.  1890. 
Albertoni,  A.     Sperimentale.     Firenze,  1883,  55. 
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Bergamo,  1877,  80.     Journ.  d'Hyg.,  Paris,  1881,  6,  255. 
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234 ;  1887,  44,  325. 
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d.  Chem.  Gesellsch,  23  ;  Kef.  27. 
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1888,  6,  177. 
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PTOMAINES.  365 

Baumann.     Zeitschr.  f.  phvsiol.  Chem.,  1,  60;  10, 123.     Schmidt's 
Jahrb.,  210,  5,  1886.   'Ber.  d.  Chem.  Gesellsch.,  9,  54,  1389, 
1715,  1747.     Vide  Udrdmzhy. 
Baumann  u.  Gergens.     Pfliiger's  Archiv,  12,  205. 
Baumert,  G.     Archiv.  d.  Pharm.  [3]  25,  911,  1887. 
Baumstark.     Zeitschr.  f.  Physiol.  Chem.,  9,  168. 
Beach,  C.  C.     New  York  Med.  Journ.,  46,  205,  1887. 
Bechamp,  A.     Theorie  Generale  de  la  Nutrition  et  Origine  des 

Ferments,  etc.     Paris,  1886. 
BecJcurts,H.     Arch.  d.  Pharm.,  220, 104, 1882.     Schmidt's  Jahrb., 

195,  9,  1882.     Chem.  Jahrb.,  1882,  1115. 
Behring.     Deutsch.  med.  Woch.,  1888,  14,  653. 
Belhj,  J.     Kozeg.  es  Torveny.  Orvos.     Budapest,  1881,  89. 
Bergeron  et  L'Hote.     Compt.    Rendus,   91,   390,    1880.     Rep.  de 

Pharm. ,  8,  458.     Archiv.  d.  Pharm.,  219,  132, 1881 ;  221,  416, 

1883. 
Bergmann.     Des  Putride  Giftund  die  Putride  Infection.     Dorpat, 

1868. 
Bergmann  u.  Schmiedeberg.     Med.  Centralbl.,  1869,  497. 
Berlinerblau.     1888. 

Berry,  J.  J.     Phila.  Times  and  Reg.,  1889,  20,  201. 
Berthelot.     Compt.  Rendus,  112,  195.     Ber.  d.  Chem.  Gesellsch., 

24,  Ref.  216 ;  22,  Ref.  700. 
Beumer  u.  Peifer.     Zeitschr.  fiir  Hygiene,  1. 
Biffi,  Seraf.     Ann.  Univ.,  223,  497,  1875. 
Billroth.     Untersuchungen  iiber  die  Vegetationsformen  von  Coc- 

cobacteria  septica,  etc.     Berlin,  1874. 
Bischoff,  F.  C.     Deutsch.  med.  Ztg.,  11,  898,  1885.    Vierteljahrschr. 

f.  gerichtl.  Med.,  1886,  44,  208. 
Blanlcenhovn.     Vide  Gamgee. 
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BoeMsch,  O.     Ber.  d.  Chem.  Gesellsch.,  18,  86,  1922;  20,  1441. 

Vide  Brieger,  Ueber  Ptomaine,  III.,  42. 
Bohm.     Arch/d.  Pharm,  224,  413,  1886.     Zeitschr.  f.  Zucker  Ind., 

13,  107.     Archiv.  f.  experim.  Pathol.,  19,  87.     Ber.  d.  Chem. 

Gesellsch.,  19,  Ref.  37. 
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Lomb.,  Milano,  1888,  43,  61. 
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Rendus,  102,  669,  727.    Lecons  sur  les  Auto-intoxications,  etc., 

Paris,  1887.    Compt.  Rendus  Soc.  d.  Biol.     Paris,  1882,  [7]  4, 

604.     Rev.  d.  Med.,  1882,  2,  825.     Med.  clin.,   Paris,  1886, 

2,  121.     France  Med.,  1886,  1,  482,  493.    Tribune  Med.,  1886, 

18,  159,  172,  195.     Union  Med.,  1886,  [3]  41,  577.     Riforma 

Med.,  Napoli,  1886,  2,  550,  556. 
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306  BIBLIOGRAPHY. 

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415,  515,  1137,  2741 ;  18, 1922;  19,  3120  ;  20,  Ref.  67,  68,  656, 
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1887. 
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Ber.  d.  Chem.  Gesellsch.,  19,  Eef.  772,  1886. 
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148,  77. 
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Gesellsch.,  21,  Eef.  756. 
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Berlin,  1886.    Annals  of  Chemical  Medicine,  1,  160-174, 1879. 

Compt.  Eeudus,  106,  1803.    Ber.  d.  Chem.  Gesellsch.,  21,  Eef. 

667. 
Tichomiroff.     Zeitschr.  f.  physiol.  Chem.,  9,  518,  566.      Ber.  d. 

Chem.  Gesellsch.,  19,  Eef.  315,  1886. 
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American  Pediatric  Society,  2,  109. 
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MlSCELLANEQUS. 


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Fliigge.     Die  Mikroorganismen. 

Fodor.     Archiv  f.  Hygiene,  4,  129. 

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Grohmann.     Ueber  die  Einwirkung  des  Zellenfreien  Blutplasma 

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386  BIBL10GEAPHY. 

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INDEX. 


ADENINE,  283 
Acleniue-hyjTOxanthine,  297 
Aerobic  bacteria,  17 
Agaricine,  237 
Albumins,  poisonous,  61 
Albumoses,  35 

immunity  from,  150 

in  urine,  355 
Alcohol,  basic  substances  in,  158 

dialysis  into,  172 

effect  on  bacterial  proteids,  173 
Alcoholic  fermentation,  bases  in,  222 
Aldehyde  collidine,  196 
Alkaloids,  interference  in    reactions 
of,  by  ptomaines,  183 

separation  from  ptomaines,  186 
Alkapton,  281 
Amanitine,  237 
American  swine- plague,  142 
Amido-valerianic  acid,  231 
Amphi-creatine,  338 
Amylarnine,  193 

Amylic  alcohol,  impurities  in,  158 
Anaerobic  bacteria,  17 
Alkaloids,  15 
Animal  chinoidine,  26,  243,  347 

coniine,  214 
Anthracin,  102,  104,  277 
Anthrax,  101 

theories  of,  85  et  seq. 

bacillus,  products  of,  101  et  seq. 

proteid,  103,  171 

albumose,  103,  156,  171 
Apricots,  poisonous,  52 
Arginine,  189,  242,  333 
Aromine,  344 
Aselline,  229 
Aseptic  fever,  361 
Asiatic  cholera,  104 

bacillus  of,  products  of,  107 
Atropine-like  substances,  27,  179 
Autogenous  diseases,  14,  352 
Azulmic  acid,  283,  288,  337 


BACILLUS  butyrieus,  16 
enteriditis,  50 
Bacon,  poisonous,  51 
Bacteria,  classification  of,  13 

in      summer      diarrhosas,     133, 
136 
Bacterial  cellular  proteids,  19 

method    of    extraction, 
130 
poisons,  definition  and  classifica- 
tion of,  15 
historical  sketch  of,  22 
foods  containing,  36 
relation    to    infectious    dis- 
eases, 84 
proteids,  18 

method  of  extraction,   170, 
171 
Bacterium  allii,  203 
Batrachians,  poison  of,  350 
Beer,    colchicine-like    substance    in, 

183 
Benzol,  impurities  in,  158 
Bergmann  and  Sehmiedeberg's  me- 
thod, 169 
Betaine,  248,  334,  343 
Bibliography    of    the    leucoma'ines, 
381 
of  the  ptomaines,  364 
Bilineurine,  237 
Biliousness,  356 
Blood,  germicidal  properties,  153 

leucomaines  in,  347 
Bocklisch's  base,  unnamed,  272 
Botulinic  acid,  46 
Bread,  poisonons,  83 
Brieger's  bases,  unnamed,  195,  255, 
261,  271,  272,  273,  274,  275 
methuds,  161 

disadvantages  of,  170 
Brouardel's  veratrine,  204 
Bujwid's  cholera-reaction,  110 
Butylamine,  193 


388 


INDEX, 


CADAVERIC  coniine,  176,  214 
Cadaverine,  34,  107,  212 
Caffeine,  316 

Canned  meats,  poisonous,  52 
Caproylamine.  194 
Carbon  monoxide  in  expired  air,  343 
Carbonic  acid,  341 
Carnine,  326,  334,  344 
Caseic  acid,  23,  46,  52 
Charcot-Neumann,  crystals  of,  330 
Cheese,  poisonous,  52 
Chemotaxis,  129 
Cholera,  104 

Bujwid's  reaction,  110 

-blue,  112 

-infantum,  133 

-red,  110 

-stools,  213 
Choline,  34,  107,237 

decompositions  of,  243 

-group, 232 

constitution  of,  252 
Chorea  gravidarum,  361 
Cicuta  virosa,  176 
Codeine-like  substances,  179 
Cod-liver  oil,  bases  from,  263 
Comma  bacillus,  ferments  produced 
by, 104 
products  of,  107  et  seq. 
Colchicine-like  substances,  181 

Zeisel's  test  for,  182 
Collidine,  28,  196,  198 
Coniine,  difficulties  in   detection  of, 
177 

-like  substances,  30, 174 
Coridine,  204 
Corindine,  202 

Corn-meal,  ptomaines  in,  33,  83,  178 
Creatine,  189,  226 
Creatinine,  189,  226 

-group, 333 
Cruso-creatinine,  336 
Cyanogen,  role  of,  336 
Cystinuria,  bases  in,  207 


DE  CONINCK'S  bases,  198,202 
Delezinier's  base,  204 
Delphinine-like  substances,  180 
Deutero-albumose,  103 

-myosinose,  156 
Dialysis,  concentration  by,  172 
Diamines,  204 
Diarrhoeas  of  infancy,  133 
Diethylamine,  191 
Digitaline-like  substances,  28, 179 


Dihydrolutidine,  195 
Dimethylamine,  34,  188 
Dimethyl-xantbine,  336 
Diphtheria,  124 

bacillus  of,  products  of,  124 

immunity  to,  128 
Dippel's  oil,  198 
Diseases,  classification  of.  84 

relation  of  bacterial  poisons  to,  84 
Dragendorffs  method,  161,  167 
Dyspepsia,  356 


E BERTH'S  bacillus,  16,  139 
products  of,  140 
Eclampsia,  362 
Eel,  jjoisonous,  41 
Ehrlich's  reagent,  260 
Enzymes,  35,  105,118 
Ethylamine,  191 
Ethyleneimine,  205,  330 
Ethylidenediamine,  34,  204     . 
Expired  air,  leucomaines  in,  341 


FECES,  poisons  in,  360 
Ferments,  35,  105,  118 
from  comma  bacillus,  104 
in  urine,  357 
Fever,  aseptic,  361 
of  exhaustion,  359 
of  fatigue,  358 
of  non-elimination,  360 
Fish,  poisonous,  41,  350 
Foods  containing  bacterial  poisons,  36 

GADININE,  258 
Gaduine,  264 
Gaduinic  acid,  263 
Galactine,  348 

Gautier's  pseudo-xanthine,  328 
Gautier  and  Etard's  bases,  199,  201, 
229 
methods,  163,  164 
extraction  of  leucomaines,  334 
German  swine-plague,  142 
Germs,  relation  of,  to  disease,  85  et  seq. 
Gerontine,  329 
Globulins,  germicidal    properties  of, 

155 
Glucosines,  223 
Glycol,  190 

Goose-grease,  poisonous,  51 
Gram's  bases,  273 
Griffith's  base,  203 


INDEX. 


389 


Guanidine,  227,  312 
Guanine,  308 
Guareschi's  base,  268 

and  Mosso's  bases,  201,  273 


HAM,  poisonous,  47 
Hankin's  method,  171 
Heteroxanthine,  319 
Hexylamine,  194 

Historical  sketch  of  the  bacterial  poi- 
sons, 22 
Hog-cholera,  142 

-erysipelas,  142 
Homo-piperidinic  acid,  231 
Hydrocollidine,  200 
Hydrocoridine,  204 
Hydrocyanic  acid,  283,  354 
Hydrolutidine,  195 
Hyoscyamine-like  substances,  27 
Hypoxanthine,  298 


ICE-CREAM,  poisonous,  79 
Immunity     from    blood     serum, 
146,  147 
methods  of  securing,  146 
-producing  substances,  nature  of 

of,  146  et  seq. 
by  intoxication  with  ptomaines, 

225 
to  diphtheria,  128 
to  pneumonia,  145 
to  swine-plague,  144 
to  tetanus,  1 19 
Indol,  111 

Infectious  diseases,  84,  101 
how  produced,  85 
definition  of,  92 
favored    by    bacterial    pro- 
ducts, 151 
Iso-amylamine,  193 
Iso-cyanacetic  acid,  351 
Iso-propylamine,  193 


K 


AKKE.  41 
Koch's  rules,  92 


LACTIC  acid,  106 
Lactockrome,  348 
Lecithin,  decomposition  of,  240 

preparation  of,  239 
Leucin,  19,  103,  109 
Leucocythfemia,  urine  in,  284 


Leucomaines,  bibliography  of,  381 

chemistry  of,  280 

extraction  of,  334 

pathological  importance  of,  354 

tables  of,  351 
Luticline,  195 
Lysatine,  189,  242,  333 
Lysatinine,  189,  242,  333 


MALIGNANT  osdema,  145 
Marino-Zuco's  method,  159 
Meal  and  bread,  poisonous,  83 
Meat,  poisonous,  50 
Methylamine,  187 

carbylamine,  351 

guanidine,  34,  108,  144,  225,  362 

hydantoin,  226,  340 

method  of  extraction,  167 

uramine,  226 

xanthine,  314,  319 
Milk,  leucomaines  in,  348 

poisonous,  62 
Monamines,  187 
Morin's  base,  222 
Morphine-like  substances,  178 
Morrhuic  acid,  263 
Morrhuine,  228 
Muscarine,  34,  251 
Mussel,  poisonous,  36 
Mutton,  poisonous,  51 
Mycoderma  aeeti,  16 
Mycoprotein,  19 
Mydatoxine,  34,  253 

isomer  of,  255,  267 
Mydaleine,  34,  270 
Mydine,  34,  230 
Mylitotoxine,  34,  40,  255 


"VTARCOTIC  substance  of  Panum,  25 
_Ll      Nencki's  base,  196 
Neuridine,  34,  218 
Neurine,  34,  232 
Nicotine-like  substances,  177 
Nicotinic  acid,  199 
Non-toxicogenic  bacteria,  13 
Nucleins,  141 


OSER'S  base,  229 
Oxy-betaines,  265 
Oxygenated  bases,  230 
Oysters,  poisonous,  41 


390 


INDEX. 


PANUM'S  narcotic  substance,  25 
putrid  poison,  24 
Paraffin  oil,  bases  in,  223 
Parareducine,  344 
Parasitic  bacteria,  13 
Paraxanthine,  321 
Parvoline,  201 
Pellagroceine,  178 
Pentamethylenediamine,  213 
Pepsin,  action  of,  357 
Peptones,   poisonous   nature   of,  354 

et  seq. 
Peptotoxine,  275,  356 
Petroleum,  bases  m,  223 
Peptotoxine,  275,  356 
Phenyl-ethylamine,  197 
Phlogosine,  274,  129 
Phosphorus-containing  substances,3 1 
Phytalbumose,  350 
Piperazine,  332 
Piperidine,  synthesis  of,  213 
Pneumonia,   chemical    products   in, 

145 
Poisonous  foods,  36 
Pouchet's  bases,  265,  268,  344 
Propylamine,  193 
Protalbumose,  103 
Protamine,  332 
Protomyosinose,  156 
Pseudo-xanthine,  328 
Ptomaines,  bibliography  of,  364 

chemistry  of,  187 

definition  of,  15 

table  of,  278,  279 

separation  of  alkaloids  from,  186 

methods  of  extraction  of,  157 
remarks  upon,  165 
Ptomatropine,  179 
Puerperal  fever,  145 
Putrefactive  alkaloids,  15 
Putrescine,  34,  107,  206 
Putrid  poison  of  Panum,  24 
Pyocyanine,  277 
Pyogenetic  proteids,  130 
Pyoxanthose,  277 
Pyridine,  107,  199,  202,  203,  275,  344 


RABBIT  septicemia,  144 
Reagents,  purity  of,  158 
Reducine,  344 
Reus's  test  for  atropine,  1 79 
Rouget,  142 
Roussin's  test  for  nicotine,  177 


O  ALAMANDARINE,  350 

kJ     Saliva,  leucoma'ines  in,  346 

Salkowski's  base,  231 

Saprophytic  bacteria,  13 

Saprine,  34,  220 

Sarcina  botulina,  46 

Sarcine,  298 

Sarcosine,  340 

Sausage,  poisonous,  22,  42 

Schweineseuche,  142 

Sebacic  acid,  22,  46,  52 

Selmi's  method,  27,  159 

Sepsine,  26 

method  of  extraction,  169 
Septicaemia  of  rabbits,  144 
Septicine,  194 
Sinapin,  242 
Spasmotoxine,  117,  194 
Spermine,  205,  330 
Spleen,  leucoma'ines  in,  347 
Staphisagria,  180 
Staphylococcus  pyog.   aureus,   bases 

from,  274 
Stas-Otto  method,  158,  167 
StercorEemia,  360 

Strychnine-like   substances,  32,  178, 
204 

reactions,  33,  180 
Sucholotoxine,  144 
Summer  diarrhoeas  of  infancy,  133 
Suppuration,  129 
Susotoxine,  143,  223 
Swine-plague,  American,  142 

products  of  bacillus  of,  143 

German,  142 


TETANINE,  34,  117,  265 
Tetanizing  substance,  32 
Tetanotoxine,  117,  194 
Tetanus,  113,  147 

bacillus,  products  of,  117  et  seq. 

immunity  to,  119 

neonatorum,  115 

toxines  194,  195,  255,265,  267 
Tetrahydronaphthylamine,  201 
Tetramethylenediamine,  209 
Tetramethyl-putrescine,  210 
Theine,  316 

Theobromine,  synthesis  of,  316 
Theophylline,  326 
Toxalbumins,  19,  35,  118,  121,  127 
Toxicogenic  bacteria,  13,  99 
Toxicology  of  ptomaines,  174 
Toxines,  15 
Toxopeptones,  109 


INDEX. 


391 


Triethylamine,  192 
Trimethylamine,  34,  189 
Trimethylenediamine,  108,  205 
Tuberculin,  120 
Tuberculosis,  120 

products  of  bacillus  of,  121 
Typhoid  bacillus,  139 

products  of,  140 

fever,  139 
Typhotoxine,  34,  140,  259 

isomer  of,  258,  261 
Tyrosin,  103,  109,197,281 
Tyrutoxicon,  34,  41,  56,  61,  79,  269 

in  summer  diarrhoea,  139 


UNDETERMINED      leucoma'ines, 
341 

ptomaines,  269  et  seq 
UrEernic  poisoning,  361 
Urea,  227 
Uric  acid,  318 

group  of  leucoma'ines,  282 
Urine,  ferments  in,  357 

leucoma'ines  in,  343 

toxicity  of,  345 


Urochrome,  344 
Urotheobromine,  34J 


VALERIANIC  acid,  231 
Vanilla,  79 
Veal,  poisonous,  51 
Venoms  of  serpents,  348 
Veratrine-like  substances,  180,  204 
Vernine,  309 

Vitiated  air,  effects  of  inhalation  of, 
362 


WEIDEL'S  reaction,  288 
White    liquefying   bacterium, 
products  of,  139 


XANTHINE,  313 
group,  constitution  of,  318 
Xantho-creatinine,  337 


z 


EISEL'S  test  for  colchicine,  182 


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